Lipids and lipid nanoparticle formulations for delivery of nucleic acids

ABSTRACT

Compounds are provided having the following structure: 
                         
or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein R 1a , R 1b , R 2a , R 2b , R 3a , R 3b , R 4a , R 4b , R 5 , R 6 , R 7 , R 8 , R 9 , L 1 , L 2 , a, b, c, d and e are as defined herein. Use of the compounds as a component of lipid nanoparticle formulations for delivery of a therapeutic agent, compositions comprising the compounds and methods for their use and preparation are also provided.

BACKGROUND

Technical Field

The present invention generally relates to novel cationic lipids thatcan be used in combination with other lipid components, such as neutrallipids, cholesterol and polymer conjugated lipids, to form lipidnanoparticles with oligonucleotides, to facilitate the intracellulardelivery of therapeutic nucleic acids (e.g. oligonucleotides, messengerRNA) both in vitro and in vivo.

Description of the Related Art

There are many challenges associated with the delivery of nucleic acidsto effect a desired response in a biological system. Nucleic acid basedtherapeutics have enormous potential but there remains a need for moreeffective delivery of nucleic acids to appropriate sites within a cellor organism in order to realize this potential. Therapeutic nucleicacids include, e.g., messenger RNA (mRNA), antisense oligonucleotides,ribozymes, DNAzymes, plasmids, immune stimulating nucleic acids,antagomir, antimir, mimic, supermir, and aptamers. Some nucleic acids,such as mRNA or plasmids, can be used to effect expression of specificcellular products as would be useful in the treatment of, for example,diseases related to a deficiency of a protein or enzyme. The therapeuticapplications of translatable nucleotide delivery are extremely broad asconstructs can be synthesized to produce any chosen protein sequence,whether or not indigenous to the system. The expression products of thenucleic acid can augment existing levels of protein, replace missing ornon-functional versions of a protein, or introduce new protein andassociated functionality in a cell or organism.

Some nucleic acids, such as miRNA inhibitors, can be used to effectexpression of specific cellular products that are regulated by miRNA aswould be useful in the treatment of, for example, diseases related todeficiency of protein or enzyme. The therapeutic applications of miRNAinhibition are extremely broad as constructs can be synthesized toinhibit one or more miRNA that would in turn regulate the expression ofmRNA products. The inhibition of endogenous miRNA can augment itsdownstream target endogenous protein expression and restore properfunction in a cell or organism as a means to treat disease associated toa specific miRNA or a group of miRNA.

Other nucleic acids can down-regulate intracellular levels of specificmRNA and, as a result, down-regulate the synthesis of the correspondingproteins through processes such as RNA interference (RNAi) orcomplementary binding of antisense RNA. The therapeutic applications ofantisense oligonucleotide and RNAi are also extremely broad, sinceoligonucleotide constructs can be synthesized with any nucleotidesequence directed against a target mRNA. Targets may include mRNAs fromnormal cells, mRNAs associated with disease-states, such as cancer, andmRNAs of infectious agents, such as viruses. To date, antisenseoligonucleotide constructs have shown the ability to specificallydown-regulate target proteins through degradation of the cognate mRNA inboth in vitro and in vivo models. In addition, antisense oligonucleotideconstructs are currently being evaluated in clinical studies.

However, two problems currently face using oligonucleotides intherapeutic contexts. First, free RNAs are susceptible to nucleasedigestion in plasma. Second, free RNAs have limited ability to gainaccess to the intracellular compartment where the relevant translationmachinery resides. Lipid nanoparticles formed from cationic lipids withother lipid components, such as neutral lipids, cholesterol, PEG,PEGylated lipids, and oligonucleotides have been used to blockdegradation of the RNAs in plasma and facilitate the cellular uptake ofthe oligonucleotides.

There remains a need for improved cationic lipids and lipidnanoparticles for the delivery of oligonucleotides. Preferably, theselipid nanoparticles would provide optimal drug:lipid ratios, protect thenucleic acid from degradation and clearance in serum, be suitable forsystemic delivery, and provide intracellular delivery of the nucleicacid. In addition, these lipid-nucleic acid particles should bewell-tolerated and provide an adequate therapeutic index, such thatpatient treatment at an effective dose of the nucleic acid is notassociated with unacceptable toxicity and/or risk to the patient. Thepresent invention provides these and related advantages.

BRIEF SUMMARY

In brief, the present invention provides lipid compounds, includingstereoisomers, pharmaceutically acceptable salts or tautomers thereof,which can be used alone or in combination with other lipid componentssuch as neutral lipids, charged lipids, steroids (including for example,all sterols) and/or their analogs, and/or polymer conjugated lipids toform lipid nanoparticles for the delivery of therapeutic agents. In someinstances, the lipid nanoparticles are used to deliver nucleic acidssuch as antisense and/or messenger RNA. Methods for use of such lipidnanoparticles for treatment of various diseases or conditions, such asthose caused by infectious entities and/or insufficiency of a protein,are also provided.

In one embodiment, compounds having the following Formula (I) areprovided:

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R^(4a), R^(4b),R⁵, R⁶, R⁷, R⁸, R⁹, L¹, L², a, b, c, d and e are as defined herein.

Pharmaceutical compositions comprising one or more of the foregoingcompounds of Formula (I) and a therapeutic agent are also provided. Insome embodiments, the pharmaceutical compositions further comprise oneor more components selected from neutral lipids, charged lipids,steroids and polymer conjugated lipids. Such compositions are useful forformation of lipid nanoparticles for the delivery of the therapeuticagent.

In other embodiments, the present invention provides a method foradministering a therapeutic agent to a patient in need thereof, themethod comprising preparing a composition of lipid nanoparticlescomprising the compound of Formula (I) and a therapeutic agent anddelivering the composition to the patient.

In still more embodiments, the invention is directed to a pegylatedlipid having the following structure (II):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein R¹⁰, R¹¹ and z are as defined herein.

These and other aspects of the invention will be apparent upon referenceto the following detailed description.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

In the figures, identical reference numbers identify similar elements.The sizes and relative positions of elements in the figures are notnecessarily drawn to scale and some of these elements are arbitrarilyenlarged and positioned to improve figure legibility. Further, theparticular shapes of the elements as drawn are not intended to conveyany information regarding the actual shape of the particular elements,and have been solely selected for ease of recognition in the figures.

FIG. 1 shows time course of luciferase expression in mouse liver.

FIG. 2 illustrates the calculation of pKa for MC3 as a representativeexample relevant to the disclosed lipids.

FIG. 3 provides comparative luciferase activity data for differentlipids.

FIG. 4 is a bar graph showing comparative data for compositionscomprising two different pegylated lipids.

DETAILED DESCRIPTION

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments of theinvention. However, one skilled in the art will understand that theinvention may be practiced without these details.

The present invention is based, in part, upon the discovery of novelcationic (amino) lipids that provide advantages when used in lipidnanoparticles for the in vivo delivery of an active or therapeutic agentsuch as a nucleic acid into a cell of a mammal. In particular,embodiments of the present invention provide nucleic acid-lipidnanoparticle compositions comprising one or more of the novel cationiclipids described herein that provide increased activity of the nucleicacid and improved tolerability of the compositions in vivo, resulting ina significant increase in the therapeutic index as compared to nucleicacid-lipid nanoparticle compositions previously described.

In particular embodiments, the present invention provides novel cationiclipids that enable the formulation of improved compositions for the invitro and in vivo delivery of mRNA and/or other oligonucleotides. Insome embodiments, these improved lipid nanoparticle compositions areuseful for expression of protein encoded by mRNA. In other embodiments,these improved lipid nanoparticles compositions are useful forupregulation of endogenous protein expression by delivering miRNAinhibitors targeting one specific miRNA or a group of miRNA regulatingone target mRNA or several mRNA. In other embodiments, these improvedlipid nanoparticle compositions are useful for down-regulating (e.g.,silencing) the protein levels and/or mRNA levels of target genes. Insome other embodiments, the lipid nanoparticles are also useful fordelivery of mRNA and plasmids for expression of transgenes. In yet otherembodiments, the lipid nanoparticle compositions are useful for inducinga pharmacological effect resulting from expression of a protein, e.gincreased production of red blood cells through the delivery of asuitable erythropoietin mRNA, or protection against infection throughdelivery of mRNA encoding for a suitable antibody.

The lipid nanoparticles and compositions of the present invention may beused for a variety of purposes, including the delivery of encapsulatedor associated (e.g., complexed) therapeutic agents such as nucleic acidsto cells, both in vitro and in vivo. Accordingly, embodiments of thepresent invention provide methods of treating or preventing diseases ordisorders in a subject in need thereof by contacting the subject with alipid nanoparticle that encapsulates or is associated with a suitabletherapeutic agent, wherein the lipid nanoparticle comprises one or moreof the novel cationic lipids described herein.

As described herein, embodiments of the lipid nanoparticles of thepresent invention are particularly useful for the delivery of nucleicacids, including, e.g., mRNA, antisense oligonucleotide, plasmid DNA,microRNA (miRNA), miRNA inhibitors (antagomirs/antimirs),messenger-RNA-interfering complementary RNA (micRNA), DNA, multivalentRNA, dicer substrate RNA, complementary DNA (cDNA), etc. Therefore, thelipid nanoparticles and compositions of the present invention may beused to induce expression of a desired protein both in vitro and in vivoby contacting cells with a lipid nanoparticle comprising one or morenovel cationic lipids described herein, wherein the lipid nanoparticleencapsulates or is associated with a nucleic acid that is expressed toproduce the desired protein (e.g., a messenger RNA or plasmid encodingthe desired protein). Alternatively, the lipid nanoparticles andcompositions of the present invention may be used to decrease theexpression of target genes and proteins both in vitro and in vivo bycontacting cells with a lipid nanoparticle comprising one or more novelcationic lipids described herein, wherein the lipid nanoparticleencapsulates or is associated with a nucleic acid that reduces targetgene expression (e.g., an antisense oligonucleotide or small interferingRNA (siRNA)). The lipid nanoparticles and compositions of the presentinvention may also be used for co-delivery of different nucleic acids(e.g. mRNA and plasmid DNA) separately or in combination, such as may beuseful to provide an effect requiring colocalization of differentnucleic acids (e.g. mRNA encoding for a suitable gene modifying enzymeand DNA segment(s) for incorporation into the host genome).

Nucleic acids for use with this invention may be prepared according toany available technique. For mRNA, the primary methodology ofpreparation is, but not limited to, enzymatic synthesis (also termed invitro transcription) which currently represents the most efficientmethod to produce long sequence-specific mRNA. In vitro transcriptiondescribes a process of template-directed synthesis of RNA molecules froman engineered DNA template comprised of an upstream bacteriophagepromoter sequence (e.g. including but not limited to that from the T7,T3 and SP6 coliphage) linked to a downstream sequence encoding the geneof interest. Template DNA can be prepared for in vitro transcriptionfrom a number of sources with appropriate techniques which are wellknown in the art including, but not limited to, plasmid DNA andpolymerase chain reaction amplification (see Linpinsel, J. L and Conn,G. L., General protocols for preparation of plasmid DNA template andBowman, J. C., Azizi, B., Lenz, T. K., Ray, P., and Williams, L. D. inRNA in vitro transcription and RNA purification by denaturing PAGE inRecombinant and in vitro RNA syntheses Methods v. 941 Conn G. L. (ed),New York, N.Y. Humana Press, 2012)

Transcription of the RNA occurs in vitro using the linearized DNAtemplate in the presence of the corresponding RNA polymerase andadenosine, guanosine, uridine and cytidine ribonucleoside triphosphates(rNTPs) under conditions that support polymerase activity whileminimizing potential degradation of the resultant mRNA transcripts. Invitro transcription can be performed using a variety of commerciallyavailable kits including, but not limited to RiboMax Large Scale RNAProduction System (Promega), MegaScript Transcription kits (LifeTechnologies) as well as with commercially available reagents includingRNA polymerases and rNTPs. The methodology for in vitro transcription ofmRNA is well known in the art. (see, e.g. Losick, R., 1972, In vitrotranscription, Ann Rev Biochem v. 41 409-46; Kamakaka, R. T. and Kraus,W. L. 2001. In Vitro Transcription. Current Protocols in Cell Biology.2:11.6:11.6.1-11.6.17; Beckert, B. And Masquida, B., (2010) Synthesis ofRNA by In Vitro Transcription in RNA in Methods in Molecular Biology v.703 (Neilson, H. Ed), New York, N.Y. Humana Press, 2010; Brunelle, J. L.and Green, R., 2013, Chapter Five—In vitro transcription from plasmid orPCR-amplified DNA, Methods in Enzymology v. 530, 101-114; all of whichare incorporated herein by reference).

The desired in vitro transcribed mRNA is then purified from theundesired components of the transcription or associated reactions(including unincorporated rNTPs, protein enzyme, salts, short RNA oligosetc). Techniques for the isolation of the mRNA transcripts are wellknown in the art. Well known procedures include phenol/chloroformextraction or precipitation with either alcohol (ethanol, isopropanol)in the presence of monovalent cations or lithium chloride. Additional,non-limiting examples of purification procedures which can be usedinclude size exclusion chromatography (Lukavsky, P. J. and Puglisi, J.D., 2004, Large-scale preparation and purification ofpolyacrylamide-free RNA oligonucleotides, RNA v. 10, 889-893),silica-based affinity chromatography and polyacrylamide gelelectrophoresis (Bowman, J. C., Azizi, B., Lenz, T. K., Ray, P., andWilliams, L. D. in RNA in vitro transcription and RNA purification bydenaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941Conn G. L. (ed), New York, N.Y. Humana Press, 2012). Purification can beperformed using a variety of commercially available kits including, butnot limited to SV Total Isolation System (Promega) and In VitroTranscription Cleanup and Concentration Kit (Norgen Biotek).

Furthermore, while reverse transcription can yield large quantities ofmRNA, the products can contain a number of aberrant RNA impuritiesassociated with undesired polymerase activity which may need to beremoved from the full-length mRNA preparation. These include short RNAsthat result from abortive transcription initiation as well asdouble-stranded RNA (dsRNA) generated by RNA-dependent RNA polymeraseactivity, RNA-primed transcription from RNA templates andself-complementary 3′ extension. It has been demonstrated that thesecontaminants with dsRNA structures can lead to undesiredimmunostimulatory activity through interaction with various innateimmune sensors in eukaryotic cells that function to recognize specificnucleic acid structures and induce potent immune responses. This inturn, can dramatically reduce mRNA translation since protein synthesisis reduced during the innate cellular immune response. Therefore,additional techniques to remove these dsRNA contaminants have beendeveloped and are known in the art including but not limited toscaleable HPLC purification (see e.g. Kariko, K., Muramatsu, H., Ludwig,J. And Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLCpurification eliminates immune activation and improves translation ofnucleoside-modified, protein-encoding mRNA, Nucl Acid Res, v. 39 e142;Weissman, D., Pardi, N., Muramatsu, H., and Kariko, K., HPLCPurification of in vitro transcribed long RNA in Synthetic Messenger RNAand Cell Metabolism Modulation in Methods in Molecular Biology v. 969(Rabinovich, P. H. Ed), 2013). HPLC purified mRNA has been reported tobe translated at much greater levels, particularly in primary cells andin vivo.

A significant variety of modifications have been described in the artwhich are used to alter specific properties of in vitro transcribedmRNA, and improve its utility. These include, but are not limited tomodifications to the 5′ and 3′ termini of the mRNA. Endogenouseukaryotic mRNA typically contain a cap structure on the 5′-end of amature molecule which plays an important role in mediating binding ofthe mRNA Cap Binding Protein (CBP), which is in turn responsible forenhancing mRNA stability in the cell and efficiency of mRNA translation.Therefore, highest levels of protein expression are achieved with cappedmRNA transcripts. The 5′-cap contains a 5′-5′-triphosphate linkagebetween the 5′-most nucleotide and guanine nucleotide. The conjugatedguanine nucleotide is methylated at the N7 position. Additionalmodifications include methylation of the ultimate and penultimate most5′-nucleotides on the 2′-hydroxyl group.

Multiple distinct cap structures can be used to generate the 5′-cap ofin vitro transcribed synthetic mRNA. 5′-capping of synthetic mRNA can beperformed co-transcriptionally with chemical cap analogs (i.e. cappingduring in vitro transcription). For example, the Anti-Reverse Cap Analog(ARCA) cap contains a 5′-5′-triphosphate guanine-guanine linkage whereone guanine contains an N7 methyl group as well as a 3′-O-methyl group.However, up to 20% of transcripts remain uncapped during thisco-transcriptional process and the synthetic cap analog is not identicalto the 5′-cap structure of an authentic cellular mRNA, potentiallyreducing translatability and cellular stability. Alternatively,synthetic mRNA molecules may also be enzymatically cappedpost-transcriptionally. These may generate a more authentic 5′-capstructure that more closely mimics, either structurally or functionally,the endogenous 5′-cap which have enhanced binding of cap bindingproteins, increased half life, reduced susceptibility to 5′endonucleases and/or reduced 5′ decapping. Numerous synthetic 5′-capanalogs have been developed and are known in the art to enhance mRNAstability and translatability (see eg. Grudzien-Nogalska, E., Kowalska,J., Su, W., Kuhn, A. N., Slepenkov, S. V., Darynkiewicz, E., Sahin, U.,Jemielity, J., and Rhoads, R. E., Synthetic mRNAs with superiortranslation and stability properties in Synthetic Messenger RNA and CellMetabolism Modulation in Methods in Molecular Biology v. 969(Rabinovich, P. H. Ed), 2013).

On the 3′-terminus, a long chain of adenine nucleotides (poly-A tail) isnormally added to mRNA molecules during RNA processing. Immediatelyafter transcription, the 3′ end of the transcript is cleaved to free a3′ hydroxyl to which poly-A polymerase adds a chain of adeninenucleotides to the RNA in a process called polyadenylation. The poly-Atail has been extensively shown to enhance both translational efficiencyand stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly (A),poly (A) binding protein and the regulation of mRNA stability, TrendsBio Sci v. 14 373-377; Guhaniyogi, J. And Brewer, G., 2001, Regulationof mRNA stability in mammalian cells, Gene, v. 265, 11-23; Dreyfus, M.And Regnier, P., 2002, The poly (A) tail of mRNAs: Bodyguard ineukaryotes, scavenger in bacteria, Cell, v. 111, 611-613).

Poly (A) tailing of in vitro transcribed mRNA can be achieved usingvarious approaches including, but not limited to, cloning of a poly (T)tract into the DNA template or by post-transcriptional addition usingPoly (A) polymerase. The first case allows in vitro transcription ofmRNA with poly (A) tails of defined length, depending on the size of thepoly (T) tract, but requires additional manipulation of the template.The latter case involves the enzymatic addition of a poly (A) tail to invitro transcribed mRNA using poly (A) polymerase which catalyzes theincorporation of adenine residues onto the 3′termini of RNA, requiringno additional manipulation of the DNA template, but results in mRNA withpoly(A) tails of heterogenous length. 5′-capping and 3′-poly (A) tailingcan be performed using a variety of commercially available kitsincluding, but not limited to Poly (A) Polymerase Tailing kit(EpiCenter), mMESSAGE mMACHINE T7 Ultra kit and Poly (A) Tailing kit(Life Technologies) as well as with commercially available reagents,various ARCA caps, Poly (A) polymerase, etc.

In addition to 5′ cap and 3′ poly adenylation, other modifications ofthe in vitro transcripts have been reported to provide benefits asrelated to efficiency of translation and stability. It is well known inthe art that pathogenic DNA and RNA can be recognized by a variety ofsensors within eukaryotes and trigger potent innate immune responses.The ability to discriminate between pathogenic and self DNA and RNA hasbeen shown to be based, at least in part, on structure and nucleosidemodifications since most nucleic acids from natural sources containmodified nucleosides In contrast, in vitro synthesized RNA lacks thesemodifications, thus rendering it immunostimulatory which in turn caninhibit effective mRNA translation as outlined above. The introductionof modified nucleosides into in vitro transcribed mRNA can be used toprevent recognition and activation of RNA sensors, thus mitigating thisundesired immunostimulatory activity and enhancing translation capacity(see eg. Kariko, K. And Weissman, D. 2007, Naturally occurringnucleoside modifications suppress the immunostimulatory activity of RNA:implication for therapeutic RNA development, Curr Opin Drug DiscovDevel, v. 10 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko,K., In vitro transcription of long RNA containing modified nucleosidesin Synthetic Messenger RNA and Cell Metabolism Modulation in Methods inMolecular Biology v. 969 (Rabinovich, P. H. Ed), 2013); Kariko, K.,Muramatsu, H., Welsh, F. A., Ludwig, J., Kato, H., Akira, S., Weissman,D., 2008, Incorporation of Pseudouridine Into mRNA Yields SuperiorNonimmunogenic Vector With Increased Translational Capacity andBiological Stability, Mol Ther v. 16, 1833-1840. The modifiednucleosides and nucleotides used in the synthesis of modified RNAs canbe prepared monitored and utilized using general methods and proceduresknown in the art. A large variety of nucleoside modifications areavailable that may be incorporated alone or in combination with othermodified nucleosides to some extent into the in vitro transcribed mRNA(see eg. US2012/0251618). In vitro synthesis of nucleoside-modified mRNAhave been reported to have reduced ability to activate immune sensorswith a concomitant enhanced translational capacity.

Other components of mRNA which can be modified to provide benefit interms of translatability and stability include the 5′ and 3′untranslated regions (UTR). Optimization of the UTRs (favorable 5′ and3′ UTRs can be obtained from cellular or viral RNAs), either both orindependently, have been shown to increase mRNA stability andtranslational efficiency of in vitro transcribed mRNA (see eg. Pardi,N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription oflong RNA containing modified nucleosides in Synthetic Messenger RNA andCell Metabolism Modulation in Methods in Molecular Biology v. 969(Rabinovich, P. H. Ed), 2013).

In addition to mRNA, other nucleic acid payloads may be used for thisinvention. For oligonucleotides, methods of preparation include but arenot limited to chemical synthesis and enzymatic, chemical cleavage of alonger precursor, in vitro transcription as described above, etc.Methods of synthesizing DNA and RNA nucleotides are widely used and wellknown in the art (see, e.g., Gait, M. J. (ed.) Oligonucleotidesynthesis: a practical approach, Oxford [Oxfordshire], Washington, D.C.:IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis:methods and applications, Methods in Molecular Biology, v. 288 (Clifton,N.J.) Totowa, N.J.: Humana Press, 2005; both of which are incorporatedherein by reference).

For plasmid DNA, preparation for use with this invention commonlyutilizes but is not limited to expansion and isolation of the plasmidDNA in vitro in a liquid culture of bacteria containing the plasmid ofinterest. The presence of a gene in the plasmid of interest that encodesresistance to a particular antibiotic (penicillin, kanamycin, etc)allows those bacteria containing the plasmid of interest to selectivegrow in antibiotic-containing cultures. Methods of isolating plasmid DNAare widely used and well known in the art (see, e.g. Heilig, J., Elbing,K. L. and Brent, R (2001) Large-Scale Preparation of Plasmid DNA.Current Protocols in Molecular Biology. 41:II:1.7:1.7.1-1.7.16; Rozkov,A., Larsson, B., Gillström, S., Björnestedt, R. and Schmidt, S. R.(2008), Large-scale production of endotoxin-free plasmids for transientexpression in mammalian cell culture. Biotechnol. Bioeng., 99: 557-566;and U.S. Pat. No. 6,197,553B1). Plasmid isolation can be performed usinga variety of commercially available kits including, but not limited toPlasmid Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo) and PureYieldMaxiPrep (Promega) kits as well as with commercially available reagents.

Various exemplary embodiments of the cationic lipids of the presentinvention, lipid nanoparticles and compositions comprising the same, andtheir use to deliver active or therapeutic agents such as nucleic acidsto modulate gene and protein expression, are described in further detailbelow.

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising” are to be construed in an open andinclusive sense, that is, as “including, but not limited to”.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. As used in the specification andclaims, the singular form “a”, “an” and “the” include plural referencesunless the context clearly dictates otherwise.

The phrase “induce expression of a desired protein” refers to theability of a nucleic acid to increase expression of the desired protein.To examine the extent of protein expression, a test sample (e.g., asample of cells in culture expressing the desired protein) or a testmammal (e.g., a mammal such as a human or an animal model such as arodent (e.g., mouse) or a non-human primate (e.g., monkey) model) iscontacted with a nucleic acid (e.g., nucleic acid in combination with alipid of the present invention). Expression of the desired protein inthe test sample or test animal is compared to expression of the desiredprotein in a control sample (e.g., a sample of cells in cultureexpressing the desired protein) or a control mammal (e.g., a mammal suchas a human or an animal model such as a rodent (e.g., mouse) ornon-human primate (e.g., monkey) model) that is not contacted with oradministered the nucleic acid. When the desired protein is present in acontrol sample or a control mammal, the expression of a desired proteinin a control sample or a control mammal may be assigned a value of 1.0.In particular embodiments, inducing expression of a desired protein isachieved when the ratio of desired protein expression in the test sampleor the test mammal to the level of desired protein expression in thecontrol sample or the control mammal is greater than 1, for example,about 1.1, 1.5, 2.0. 5.0 or 10.0. When a desired protein is not presentin a control sample or a control mammal, inducing expression of adesired protein is achieved when any measurable level of the desiredprotein in the test sample or the test mammal is detected. One ofordinary skill in the art will understand appropriate assays todetermine the level of protein expression in a sample, for example dotblots, northern blots, in situ hybridization, ELISA,immunoprecipitation, enzyme function, and phenotypic assays, or assaysbased on reporter proteins that can produce fluorescence or luminescenceunder appropriate conditions.

The phrase “inhibiting expression of a target gene” refers to theability of a nucleic acid to silence, reduce, or inhibit the expressionof a target gene. To examine the extent of gene silencing, a test sample(e.g., a sample of cells in culture expressing the target gene) or atest mammal (e.g., a mammal such as a human or an animal model such as arodent (e.g., mouse) or a non-human primate (e.g., monkey) model) iscontacted with a nucleic acid that silences, reduces, or inhibitsexpression of the target gene. Expression of the target gene in the testsample or test animal is compared to expression of the target gene in acontrol sample (e.g., a sample of cells in culture expressing the targetgene) or a control mammal (e.g., a mammal such as a human or an animalmodel such as a rodent (e.g., mouse) or non-human primate (e.g., monkey)model) that is not contacted with or administered the nucleic acid. Theexpression of the target gene in a control sample or a control mammalmay be assigned a value of 100%. In particular embodiments, silencing,inhibition, or reduction of expression of a target gene is achieved whenthe level of target gene expression in the test sample or the testmammal relative to the level of target gene expression in the controlsample or the control mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%,60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Inother words, the nucleic acids are capable of silencing, reducing, orinhibiting the expression of a target gene by at least about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% in a test sample or a test mammal relative to thelevel of target gene expression in a control sample or a control mammalnot contacted with or administered the nucleic acid. Suitable assays fordetermining the level of target gene expression include, withoutlimitation, examination of protein or mRNA levels using techniques knownto those of skill in the art, such as, e.g., dot blots, northern blots,in situ hybridization, ELISA, immunoprecipitation, enzyme function, aswell as phenotypic assays known to those of skill in the art.

An “effective amount” or “therapeutically effective amount” of an activeagent or therapeutic agent such as a therapeutic nucleic acid is anamount sufficient to produce the desired effect, e.g., an increase orinhibition of expression of a target sequence in comparison to thenormal expression level detected in the absence of the nucleic acid. Anincrease in expression of a target sequence is achieved when anymeasurable level is detected in the case of an expression product thatis not present in the absence of the nucleic acid. In the case where theexpression product is present at some level prior to contact with thenucleic acid, an in increase in expression is achieved when the foldincrease in value obtained with a nucleic acid such as mRNA relative tocontrol is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000,5000, 10000 or greater. Inhibition of expression of a target gene ortarget sequence is achieved when the value obtained with a nucleic acidsuch as antisense oligonucleotide relative to the control is about 95%,90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%,20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring expression of atarget gene or target sequence include, e.g., examination of protein orRNA levels using techniques known to those of skill in the art such asdot blots, northern blots, in situ hybridization, ELISA,immunoprecipitation, enzyme function, fluorescence or luminescence ofsuitable reporter proteins, as well as phenotypic assays known to thoseof skill in the art.

The term “nucleic acid” as used herein refers to a polymer containing atleast two deoxyribonucleotides or ribonucleotides in either single- ordouble-stranded form and includes DNA, RNA, and hybrids thereof. DNA maybe in the form of antisense molecules, plasmid DNA, cDNA, PCR products,or vectors. RNA may be in the form of small hairpin RNA (shRNA),messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA,dicer substrate RNA or viral RNA (vRNA), and combinations thereof.Nucleic acids include nucleic acids containing known nucleotide analogsor modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, and which have similarbinding properties as the reference nucleic acid. Examples of suchanalogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2′-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions), alleles, orthologs, single nucleotide polymorphisms, andcomplementary sequences as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka etal., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell.Probes, 8:91-98 (1994)). “Nucleotides” contain a sugar deoxyribose (DNA)or ribose (RNA), a base, and a phosphate group. Nucleotides are linkedtogether through the phosphate groups. “Bases” include purines andpyrimidines, which further include natural compounds adenine, thymine,guanine, cytosine, uracil, inosine, and natural analogs, and syntheticderivatives of purines and pyrimidines, which include, but are notlimited to, modifications which place new reactive groups such as, butnot limited to, amines, alcohols, thiols, carboxylates, andalkylhalides.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises partial length or entire length coding sequencesnecessary for the production of a polypeptide or precursor polypeptide.

“Gene product,” as used herein, refers to a product of a gene such as anRNA transcript or a polypeptide.

The term “lipid” refers to a group of organic compounds that include,but are not limited to, esters of fatty acids and are generallycharacterized by being poorly soluble in water, but soluble in manyorganic solvents. They are usually divided into at least three classes:(1) “simple lipids,” which include fats and oils as well as waxes; (2)“compound lipids,” which include phospholipids and glycolipids; and (3)“derived lipids” such as steroids.

A “steroid” is a compound comprising the following carbon skeleton:

Non-limiting examples of steroids include cholesterol, and the like.

A “cationic lipid” refers to a lipid capable of being positivelycharged. Exemplary cationic lipids include one or more amine group(s)which bear the positive charge. Preferred cationic lipids are ionizablesuch that they can exist in a positively charged or neutral formdepending on pH. The ionization of the cationic lipid affects thesurface charge of the lipid nanoparticle under different pH conditions.This charge state can influence plasma protein absorption, bloodclearance and tissue distribution (Semple, S. C., et al., Adv. DrugDeliv Rev 32:3-17 (1998)) as well as the ability to form endosomolyticnon-bilayer structures (Hafez, I. M., et al., Gene Ther 8:1188-1196(2001)) critical to the intracellular delivery of nucleic acids.

The term “lipid nanoparticle” refers to particles having at least onedimension on the order of nanometers (e.g., 1-1,000 nm) which includeone or more of the compounds of formula (I) or other specified cationiclipids. In some embodiments, lipid nanoparticles are included in aformulation that can be used to deliver an active agent or therapeuticagent, such as a nucleic acid (e.g., mRNA) to a target site of interest(e.g., cell, tissue, organ, tumor, and the like). In some embodiments,the lipid nanoparticles of the invention comprise a nucleic acid. Suchlipid nanoparticles typically comprise a compound of Formula (I) and oneor more excipient selected from neutral lipids, charged lipids, steroidsand polymer conjugated lipids. In some embodiments, the active agent ortherapeutic agent, such as a nucleic acid, may be encapsulated in thelipid portion of the lipid nanoparticle or an aqueous space enveloped bysome or all of the lipid portion of the lipid nanoparticle, therebyprotecting it from enzymatic degradation or other undesirable effectsinduced by the mechanisms of the host organism or cells e.g. an adverseimmune response.

In various embodiments, the lipid nanoparticles have a mean diameter offrom about 30 nm to about 150 nm, from about 40 nm to about 150 nm, fromabout 50 nm to about 150 nm, from about 60 nm to about 130 nm, fromabout 70 nm to about 110 nm, from about 70 nm to about 100 nm, fromabout 80 nm to about 100 nm, from about 90 nm to about 100 nm, fromabout 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm,and are substantially non-toxic. In certain embodiments, nucleic acids,when present in the lipid nanoparticles, are resistant in aqueoussolution to degradation with a nuclease. Lipid nanoparticles comprisingnucleic acids and their method of preparation are disclosed in, e.g.,U.S. Patent Publication Nos. 2004/0142025, 2007/0042031 and PCT Pub.Nos. WO 2013/016058 and WO 2013/086373, the full disclosures of whichare herein incorporated by reference in their entirety for all purposes.

As used herein, “lipid encapsulated” refers to a lipid nanoparticle thatprovides an active agent or therapeutic agent, such as a nucleic acid(e.g., mRNA), with full encapsulation, partial encapsulation, or both.In an embodiment, the nucleic acid (e.g., mRNA) is fully encapsulated inthe lipid nanoparticle.

The term “polymer conjugated lipid” refers to a molecule comprising botha lipid portion and a polymer portion. An example of a polymerconjugated lipid is a pegylated lipid. The term “pegylated lipid” refersto a molecule comprising both a lipid portion and a polyethylene glycolportion. Pegylated lipids are known in the art and include1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG) andthe like.

The term “neutral lipid” refers to any of a number of lipid species thatexist either in an uncharged or neutral zwitterionic form at a selectedpH. At physiological pH, such lipids include, but are not limited to,phosphotidylcholines such as 1,2-Distearoyl-sn-glycero-3-phosphocholine(DSPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),phophatidylethanolamines such as1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelins(SM), ceramides, steroids such as sterols and their derivatives. Neutrallipids may be synthetic or naturally derived.

The term “charged lipid” refers to any of a number of lipid species thatexist in either a positively charged or negatively charged formindependent of the pH within a useful physiological range e.g. pH ˜3 topH ˜9. Charged lipids may be synthetic or naturally derived. Examples ofcharged lipids include phosphatidylserines, phosphatidic acids,phosphatidylglycerols, phosphatidylinositols, sterol hemisuccinates,dialkyl trimethylammonium-propanes, (e.g. DOTAP, DOTMA), dialkyldimethylaminopropanes, ethyl phosphocholines, dimethylaminoethanecarbamoyl sterols (e.g. DC-Chol).

As used herein, the term “aqueous solution” refers to a compositioncomprising water.

“Serum-stable” in relation to nucleic acid-lipid nanoparticles meansthat the nucleotide is not significantly degraded after exposure to aserum or nuclease assay that would significantly degrade free DNA orRNA. Suitable assays include, for example, a standard serum assay, aDNAse assay, or an RNAse assay.

“Systemic delivery,” as used herein, refers to delivery of a therapeuticproduct that can result in a broad exposure of an active agent within anorganism. Some techniques of administration can lead to the systemicdelivery of certain agents, but not others. Systemic delivery means thata useful, preferably therapeutic, amount of an agent is exposed to mostparts of the body. Systemic delivery of lipid nanoparticles can be byany means known in the art including, for example, intravenous,intraarterial, subcutaneous, and intraperitoneal delivery. In someembodiments, systemic delivery of lipid nanoparticles is by intravenousdelivery.

“Local delivery,” as used herein, refers to delivery of an active agentdirectly to a target site within an organism. For example, an agent canbe locally delivered by direct injection into a disease site such as atumor, other target site such as a site of inflammation, or a targetorgan such as the liver, heart, pancreas, kidney, and the like. Localdelivery can also include topical applications or localized injectiontechniques such as intramuscular, subcutaneous or intradermal injection.Local delivery does not preclude a systemic pharmacological effect.

“Alkyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, which is saturated orunsaturated (i.e., contains one or more double and/or triple bonds),having from one to twenty-four carbon atoms (C₁-C₂₄ alkyl), one totwelve carbon atoms (C₁-C₁₂ alkyl), one to eight carbon atoms (C₁-C₈alkyl) or one to six carbon atoms (C₁-C₆ alkyl) and which is attached tothe rest of the molecule by a single bond, e.g., methyl, ethyl, npropyl, 1 methylethyl (iso propyl), n butyl, n pentyl, 1,1 dimethylethyl(t butyl), 3 methylhexyl, 2 methylhexyl, ethenyl, prop 1 enyl, but 1enyl, pent 1 enyl, penta 1,4 dienyl, ethynyl, propynyl, butynyl,pentynyl, hexynyl, and the like. Unless stated otherwise specifically inthe specification, an alkyl group is optionally substituted.

“Cycloalkyl” or “carbocyclic ring” refers to a stable non aromaticmonocyclic or polycyclic hydrocarbon radical consisting solely of carbonand hydrogen atoms, which may include fused or bridged ring systems,having from three to fifteen carbon atoms, preferably having from threeto ten carbon atoms, and which is saturated or unsaturated and attachedto the rest of the molecule by a single bond. Monocyclic radicalsinclude, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example,adamantyl, norbornyl, decalinyl, 7,7 dimethyl bicyclo[2.2.1]heptanyl,and the like. Unless otherwise stated specifically in the specification,a cycloalkyl group is optionally substituted.

“Heterocyclyl” or “heterocyclic ring” refers to a stable 3- to18-membered non-aromatic ring radical which consists of two to twelvecarbon atoms and from one to six heteroatoms selected from the groupconsisting of nitrogen, oxygen and sulfur. Unless stated otherwisespecifically in the specification, the heterocyclyl radical may be amonocyclic, bicyclic, tricyclic or tetracyclic ring system, which mayinclude fused or bridged ring systems; and the nitrogen, carbon orsulfur atoms in the heterocyclyl radical may be optionally oxidized; thenitrogen atom may be optionally quaternized; and the heterocyclylradical may be partially or fully saturated. Examples of suchheterocyclyl radicals include, but are not limited to, dioxolanyl,thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl,imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl,octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl,piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl,thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl,thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in thespecification, a heterocyclyl group may be optionally substituted.

The term “substituted” used herein means any of the above groups (e.g.,alkyl, cycloalkyl or heterocyclyl) wherein at least one hydrogen atom isreplaced by a bond to a non-hydrogen atoms such as, but not limited to:a halogen atom such as F, Cl, Br, and I; oxo groups (═O); hydroxylgroups (—OH); alkoxy groups (—OR^(a), where R^(a) is C₁-C₁₂ alkyl orcycloalkyl); carboxyl groups (—OC(═O)R^(a) or —C(═O)OR^(a), where R^(a)is H, C₁-C₁₂ alkyl or cycloalkyl); amine groups (—NR^(a)R^(b), whereR^(a) and R^(b) are each independently H, C₁-C₁₂ alkyl or cycloalkyl);C₁-C₁₂ alkyl groups; and cycloalkyl groups. In some embodiments thesubstituent is a C₁-C₁₂ alkyl group. In other embodiments, thesubstituent is a cycloalkyl group. In other embodiments, the substituentis a halo group, such as fluoro. In other embodiments, the substituentis a oxo group. In other embodiments, the substituent is a hydroxylgroup. In other embodiments, the substituent is an alkoxy group. Inother embodiments, the substituent is a carboxyl group. In otherembodiments, the substituent is an amine group.

“Optional” or “optionally” (e.g., optionally substituted) means that thesubsequently described event of circumstances may or may not occur, andthat the description includes instances where said event or circumstanceoccurs and instances in which it does not. For example, “optionallysubstituted alkyl” means that the alkyl radical may or may not besubstituted and that the description includes both substituted alkylradicals and alkyl radicals having no substitution.

“Prodrug” is meant to indicate a compound that may be converted underphysiological conditions or by solvolysis to a biologically activecompound of the invention. Thus, the term “prodrug” refers to ametabolic precursor of a compound of the invention that ispharmaceutically acceptable. A prodrug may be inactive when administeredto a subject in need thereof, but is converted in vivo to an activecompound of the invention. Prodrugs are typically rapidly transformed invivo to yield the parent compound of the invention, for example, byhydrolysis in blood. The prodrug compound often offers advantages ofsolubility, tissue compatibility or delayed release in a mammalianorganism (see, Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24(Elsevier, Amsterdam)). A discussion of prodrugs is provided in Higuchi,T., et al., A.C.S. Symposium Series, Vol. 14, and in BioreversibleCarriers in Drug Design, Ed. Edward B. Roche, American PharmaceuticalAssociation and Pergamon Press, 1987.

The term “prodrug” is also meant to include any covalently bondedcarriers, which release the active compound of the invention in vivowhen such prodrug is administered to a mammalian subject. Prodrugs of acompound of the invention may be prepared by modifying functional groupspresent in the compound of the invention in such a way that themodifications are cleaved, either in routine manipulation or in vivo, tothe parent compound of the invention. Prodrugs include compounds of theinvention wherein a hydroxy, amino or mercapto group is bonded to anygroup that, when the prodrug of the compound of the invention isadministered to a mammalian subject, cleaves to form a free hydroxy,free amino or free mercapto group, respectively. Examples of prodrugsinclude, but are not limited to, acetate, formate and benzoatederivatives of alcohol or amide derivatives of amine functional groupsin the compounds of the invention and the like.

The invention disclosed herein is also meant to encompass allpharmaceutically acceptable compounds of the compound of Formula (I) or(II) being isotopically-labelled by having one or more atoms replaced byan atom having a different atomic mass or mass number. Examples ofisotopes that can be incorporated into the disclosed compounds includeisotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine,chlorine, and iodine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O,¹⁸O, ³¹P, ³²P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²³I, and ¹²⁵I, respectively. Theseradiolabelled compounds could be useful to help determine or measure theeffectiveness of the compounds, by characterizing, for example, the siteor mode of action, or binding affinity to pharmacologically importantsite of action. Certain isotopically-labelled compounds of structure (I)or (II), for example, those incorporating a radioactive isotope, areuseful in drug and/or substrate tissue distribution studies. Theradioactive isotopes tritium, i.e., ³H, and carbon-14, i.e., ¹⁴C, areparticularly useful for this purpose in view of their ease ofincorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium, i.e., ²H, mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, can be useful in Positron Emission Topography (PET) studies forexamining substrate receptor occupancy. Isotopically-labeled compoundsof structure (I) or (II) can generally be prepared by conventionaltechniques known to those skilled in the art or by processes analogousto those described in the Preparations and Examples as set out belowusing an appropriate isotopically-labeled reagent in place of thenon-labeled reagent previously employed.

The invention disclosed herein is also meant to encompass the in vivometabolic products of the disclosed compounds. Such products may resultfrom, for example, the oxidation, reduction, hydrolysis, amidation,esterification, and the like of the administered compound, primarily dueto enzymatic processes. Accordingly, the invention includes compoundsproduced by a process comprising administering a compound of thisinvention to a mammal for a period of time sufficient to yield ametabolic product thereof. Such products are typically identified byadministering a radiolabelled compound of the invention in a detectabledose to an animal, such as rat, mouse, guinea pig, monkey, or to human,allowing sufficient time for metabolism to occur, and isolating itsconversion products from the urine, blood or other biological samples.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

“Mammal” includes humans and both domestic animals such as laboratoryanimals and household pets (e.g., cats, dogs, swine, cattle, sheep,goats, horses, rabbits), and non-domestic animals such as wildlife andthe like.

“Pharmaceutically acceptable carrier, diluent or excipient” includeswithout limitation any adjuvant, carrier, excipient, glidant, sweeteningagent, diluent, preservative, dye/colorant, flavor enhancer, surfactant,wetting agent, dispersing agent, suspending agent, stabilizer, isotonicagent, solvent, or emulsifier which has been approved by the UnitedStates Food and Drug Administration as being acceptable for use inhumans or domestic animals.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as, but are not limited to,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as, but not limitedto, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid,ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid,4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid,citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonicacid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid,fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,gluconic acid, glucuronic acid, glutamic acid, glutaric acid,2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuricacid, isobutyric acid, lactic acid, lactobionic acid, lauric acid,maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonicacid, mucic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid,propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid,4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid,tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroaceticacid, undecylenic acid, and the like.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.Preferred inorganic salts are the ammonium, sodium, potassium, calcium,and magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as ammonia,isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

Often crystallizations produce a solvate of the compound of theinvention. As used herein, the term “solvate” refers to an aggregatethat comprises one or more molecules of a compound of the invention withone or more molecules of solvent. The solvent may be water, in whichcase the solvate may be a hydrate. Alternatively, the solvent may be anorganic solvent. Thus, the compounds of the present invention may existas a hydrate, including a monohydrate, dihydrate, hemihydrate,sesquihydrate, trihydrate, tetrahydrate and the like, as well as thecorresponding solvated forms. The compound of the invention may be truesolvates, while in other cases, the compound of the invention may merelyretain adventitious water or be a mixture of water plus someadventitious solvent.

A “pharmaceutical composition” refers to a formulation of a compound ofthe invention and a medium generally accepted in the art for thedelivery of the biologically active compound to mammals, e.g., humans.Such a medium includes all pharmaceutically acceptable carriers,diluents or excipients therefor.

“Effective amount” or “therapeutically effective amount” refers to thatamount of a compound of the invention which, when administered to amammal, preferably a human, is sufficient to effect treatment in themammal, preferably a human. The amount of a lipid nanoparticle of theinvention which constitutes a “therapeutically effective amount” willvary depending on the compound, the condition and its severity, themanner of administration, and the age of the mammal to be treated, butcan be determined routinely by one of ordinary skill in the art havingregard to his own knowledge and to this disclosure.

“Treating” or “treatment” as used herein covers the treatment of thedisease or condition of interest in a mammal, preferably a human, havingthe disease or condition of interest, and includes:

(i) preventing the disease or condition from occurring in a mammal, inparticular, when such mammal is predisposed to the condition but has notyet been diagnosed as having it;

(ii) inhibiting the disease or condition, i.e., arresting itsdevelopment;

(iii) relieving the disease or condition, i.e., causing regression ofthe disease or condition; or

(iv) relieving the symptoms resulting from the disease or condition,i.e., relieving pain without addressing the underlying disease orcondition. As used herein, the terms “disease” and “condition” may beused interchangeably or may be different in that the particular maladyor condition may not have a known causative agent (so that etiology hasnot yet been worked out) and it is therefore not yet recognized as adisease but only as an undesirable condition or syndrome, wherein a moreor less specific set of symptoms have been identified by clinicians.

The compounds of the invention, or their pharmaceutically acceptablesalts may contain one or more asymmetric centers and may thus give riseto enantiomers, diastereomers, and other stereoisomeric forms that maybe defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as(D)- or (L)- for amino acids. The present invention is meant to includeall such possible isomers, as well as their racemic and optically pureforms. Optically active (+) and (−), (R)- and (S)-, or (D)- and(L)-isomers may be prepared using chiral synthons or chiral reagents, orresolved using conventional techniques, for example, chromatography andfractional crystallization. Conventional techniques for thepreparation/isolation of individual enantiomers include chiral synthesisfrom a suitable optically pure precursor or resolution of the racemate(or the racemate of a salt or derivative) using, for example, chiralhigh pressure liquid chromatography (HPLC). When the compounds describedherein contain olefinic double bonds or other centers of geometricasymmetry, and unless specified otherwise, it is intended that thecompounds include both E and Z geometric isomers. Likewise, alltautomeric forms are also intended to be included.

A “stereoisomer” refers to a compound made up of the same atoms bondedby the same bonds but having different three-dimensional structures,which are not interchangeable. The present invention contemplatesvarious stereoisomers and mixtures thereof and includes “enantiomers”,which refers to two stereoisomers whose molecules are nonsuperimposeablemirror images of one another.

A “tautomer” refers to a proton shift from one atom of a molecule toanother atom of the same molecule. The present invention includestautomers of any said compounds.

Compounds

In an aspect, the invention provides novel lipid compounds which arecapable of combining with other lipid components such as neutral lipids,charged lipids, steroids and/or polymer conjugated-lipids to form lipidnanoparticles with oligonucleotides. Without wishing to be bound bytheory, it is thought that these lipid nanoparticles shieldoligonucleotides from degradation in the serum and provide for effectivedelivery of oligonucleotides to cells in vitro and in vivo.

In one embodiment, the lipid compounds have the structure of Formula(I):

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein:

L¹ and L² are each independently —O(C═O)—, —(C═O)O— or a carbon-carbondouble bond;

R^(1a) and R^(1b) are, at each occurrence, independently either (a) H orC₁-C₁₂ alkyl, or (b) R^(1a) is H or C₁-C₁₂ alkyl, and R^(1b) togetherwith the carbon atom to which it is bound is taken together with anadjacent R^(1b) and the carbon atom to which it is bound to form acarbon-carbon double bond;

R^(2a) and R^(2b) are, at each occurrence, independently either (a) H orC₁-C₁₂ alkyl, or (b) R^(2a) is H or C₁-C₁₂ alkyl, and R^(2b) togetherwith the carbon atom to which it is bound is taken together with anadjacent R^(2b) and the carbon atom to which it is bound to form acarbon-carbon double bond;

R^(3a) and R^(3b) are, at each occurrence, independently either (a) H orC₁-C₁₂ alkyl, or (b) R^(3a) is H or C₁-C₁₂ alkyl, and R^(3b) togetherwith the carbon atom to which it is bound is taken together with anadjacent R^(3b) and the carbon atom to which it is bound to form acarbon-carbon double bond;

R^(4a) and R^(4b) are, at each occurrence, independently either (a) H orC₁-C₁₂ alkyl, or (b) R^(4a) is H or C₁-C₁₂ alkyl, and R^(4b) togetherwith the carbon atom to which it is bound is taken together with anadjacent R^(4b) and the carbon atom to which it is bound to form acarbon-carbon double bond;

R⁵ and R⁶ are each independently methyl or cycloalkyl;

R⁷ is, at each occurrence, independently H or C₁-C₁₂ alkyl;

R⁸ and R⁹ are each independently unsubstituted C₁-C₁₂ alkyl; or R⁸ andR⁹, together with the nitrogen atom to which they are attached, form a5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom;

a and d are each independently an integer from 0 to 24;

b and c are each independently an integer from 1 to 24; and

e is 1 or 2.

In certain embodiments of the Formula (I) compound at least one ofR^(1a), R^(2a), R^(3a) or R^(4a) is C₁-C₁₂ alkyl, or at least one of L¹or L² is —O(C═O)— or —(C═O)O—. In other embodiments, R^(1a) and R^(1b)are not isopropyl when a is 6 or n-butyl when a is 8.

In still further embodiments, at least one of R^(1a), R^(2a), R^(3a) orR^(4a) is C₁-C₁₂ alkyl, or at least one of L¹ or L² is —O(C═O)— or—(C═O)O—; and

R^(1a) and R^(1b) are not isopropyl when a is 6 or n-butyl when a is 8

In the compound of Formula I, any one of L¹ or L² may be —O(C═O)— or acarbon-carbon double bond. L¹ and L² may each be —O(C═O)— or may each bea carbon-carbon double bond.

In some embodiments, one of L¹ or L² is —O(C═O)—. In other embodiments,both L¹ and L² are —O(C═O)—.

In some embodiments, one of L¹ or L² is —(C═O)O—. In other embodiments,both L¹ and L² are —(C═O)O—.

In some embodiments, one of L¹ or L² is a carbon-carbon double bond. Inother embodiments, both L¹ and L² are a carbon-carbon double bond.

In still other embodiments, one of L¹ or L² is —O(C═O)— and the other ofL¹ or L² is —(C═O)O—. In more embodiments, one of L¹ or L² is —O(C═O)—and the other of L¹ or L² is a carbon-carbon double bond. In yet moreembodiments, one of L¹ or L² is —(C═O)O— and the other of L¹ or L² is acarbon-carbon double bond.

It is understood that “carbon-carbon” double bond refers to one of thefollowing structures:

wherein R^(a) and R^(b) are, at each occurrence, independently H or asubstituent. For example, in some embodiments R^(a) and R^(b) are, ateach occurrence, independently H, C₁-C₁₂ alkyl or cycloalkyl, forexample H or C₁-C₁₂ alkyl.

In other embodiments, the lipid compounds have the following structure(Ia):

In other embodiments, the lipid compounds have the following structure(Ib):

In yet other embodiments, the lipid compounds have the followingstructure (Ic):

In certain embodiments of the foregoing, a, b, c and d are eachindependently an integer from 2 to 12 or an integer from 4 to 12. Inother embodiments, a, b, c and d are each independently an integer from8 to 12 or 5 to 9. In some certain embodiments, a is 0. In someembodiments, a is 1. In other embodiments, a is 2. In more embodiments,a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5.In other embodiments, a is 6. In more embodiments, a is 7. In yet otherembodiments, a is 8. In some embodiments, a is 9. In other embodiments,a is 10. In more embodiments, a is 11. In yet other embodiments, a is12. In some embodiments, a is 13. In other embodiments, a is 14. In moreembodiments, a is 15. In yet other embodiments, a is 16.

In some embodiments, b is 1. In other embodiments, b is 2. In moreembodiments, b is 3. In yet other embodiments, b is 4. In someembodiments, b is 5. In other embodiments, b is 6. In more embodiments,b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9.In other embodiments, b is 10. In more embodiments, b is 11. In yetother embodiments, b is 12. In some embodiments, b is 13. In otherembodiments, b is 14. In more embodiments, b is 15. In yet otherembodiments, b is 16.

In some embodiments, c is 1. In other embodiments, c is 2. In moreembodiments, c is 3. In yet other embodiments, c is 4. In someembodiments, c is 5. In other embodiments, c is 6. In more embodiments,c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9.In other embodiments, c is 10. In more embodiments, c is 11. In yetother embodiments, c is 12. In some embodiments, c is 13. In otherembodiments, c is 14. In more embodiments, c is 15. In yet otherembodiments, c is 16.

In some certain embodiments, d is 0. In some embodiments, d is 1. Inother embodiments, d is 2. In more embodiments, d is 3. In yet otherembodiments, d is 4. In some embodiments, d is 5. In other embodiments,d is 6. In more embodiments, d is 7. In yet other embodiments, d is 8.In some embodiments, d is 9. In other embodiments, d is 10. In moreembodiments, d is 11. In yet other embodiments, d is 12. In someembodiments, d is 13. In other embodiments, d is 14. In moreembodiments, d is 15. In yet other embodiments, d is 16.

In some other various embodiments, a and d are the same. In some otherembodiments, b and c are the same. In some other specific embodimentsand a and d are the same and b and c are the same.

The sum of a and b and the sum of c and d are factors which may bevaried to obtain a lipid having the desired properties. In oneembodiment, a and b are chosen such that their sum is an integer rangingfrom 14 to 24. In other embodiments, c and d are chosen such that theirsum is an integer ranging from 14 to 24. In further embodiment, the sumof a and b and the sum of c and d are the same. For example, in someembodiments the sum of a and b and the sum of c and d are both the sameinteger which may range from 14 to 24. In still more embodiments, a. b,c and d are selected such the sum of a and b and the sum of c and d is12 or greater.

In some embodiments, e is 1. In other embodiments, e is 2.

The substituents at R^(1a), R^(2a), R^(3a) and R^(4a) are notparticularly limited. In certain embodiments R^(1a), R^(2a), R^(3a) andR^(4a) are H at each occurrence. In certain other embodiments at leastone of R^(1a), R^(2a), R^(3a) and R^(4a) is C₁-C₁₂ alkyl. In certainother embodiments at least one of R^(1a), R^(2a), R^(3a) and R^(4a) isC₁-C₈ alkyl. In certain other embodiments at least one of R^(1a),R^(2a), R^(3a) and R^(4a) is C₁-C₆ alkyl. In some of the foregoingembodiments, the C₁-C₈ alkyl is methyl, ethyl, n-propyl, iso-propyl,n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

In certain embodiments of the foregoing, R^(1a), R^(1b), R^(4a) andR^(4b) are C₁-C₁₂ alkyl at each occurrence.

In further embodiments of the foregoing, at least one of R^(1b), R^(2b),R^(3b) and R^(4b) is H or R^(1b), R^(2b), R^(3b) and R^(4b) are H ateach occurrence.

In certain embodiments of the foregoing, R^(1b) together with the carbonatom to which it is bound is taken together with an adjacent R^(1b) andthe carbon atom to which it is bound to form a carbon-carbon doublebond. In other embodiments of the foregoing R^(4b) together with thecarbon atom to which it is bound is taken together with an adjacentR^(4b) and the carbon atom to which it is bound to form a carbon-carbondouble bond.

The substituents at R⁵ and R⁶ are not particularly limited in theforegoing embodiments. In certain embodiments one or both of R⁵ or R⁶ ismethyl. In certain other embodiments one or both of R⁵ or R⁶ iscycloalkyl for example cyclohexyl. In these embodiments the cycloalkylmay be substituted or not substituted. In certain other embodiments thecycloalkyl is substituted with C₁-C₁₂ alkyl, for example tert-butyl.

The substituents at R⁷ are not particularly limited in the foregoingembodiments. In certain embodiments at least one R⁷ is H. In some otherembodiments, R⁷ is H at each occurrence. In certain other embodiments R⁷is C₁-C₁₂ alkyl.

In certain other of the foregoing embodiments, one of R⁸ or R⁹ ismethyl. In other embodiments, both R⁸ and R⁹ are methyl.

In some different embodiments, R⁸ and R⁹, together with the nitrogenatom to which they are attached, form a 5, 6 or 7-membered heterocyclicring. In some embodiments of the foregoing, R⁸ and R⁹, together with thenitrogen atom to which they are attached, form a 5-membered heterocyclicring, for example a pyrrolidinyl ring.

In various different embodiments, the compound has one of the structuresset forth in Table 1 below.

TABLE 1 Representative Compounds Preparation No. Structure Method 1

B 2

A 3

A 4

B 5

B 6

B 7

A 8

A 9

B 10

A 11

A 12

A 13

A 14

A 15

A 16

A 17

A 18

A 19

A 20

A 21

A 22

A 23

A 24

A 25

A 26

A 27

A 28

A 29

A 30

A 31

C 32

C 33

C 34

B 35

B 36

C 37

C 38

B 39

B 40

B 41

B

It is understood that any embodiment of the compounds of Formula (I), asset forth above, and any specific substituent and/or variable in thecompound Formula (I), as set forth above, may be independently combinedwith other embodiments and/or substituents and/or variables of compoundsof Formula (I) to form embodiments of the inventions not specificallyset forth above. In addition, in the event that a list of substituentsand/or variables is listed for any particular R group, L group orvariables a-e in a particular embodiment and/or claim, it is understoodthat each individual substituent and/or variable may be deleted from theparticular embodiment and/or claim and that the remaining list ofsubstituents and/or variables will be considered to be within the scopeof the invention.

It is understood that in the present description, combinations ofsubstituents and/or variables of the depicted formulae are permissibleonly if such contributions result in stable compounds.

In various embodiments, it is understood that the following compoundsare not included within the scope of the invention:

wherein each R^(c) and R^(d) are H or R^(c) and R^(d) join to form oxo,and x and y are each independently an integer from 0 to 6.

Also provided in various embodiments are pegylated lipids. For examplein an embodiment, the pegylated lipid has the following structure (II):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein:

R¹⁰ and R¹¹ are each independently a straight or branched, saturated orunsaturated alkyl chain containing from 10 to 30 carbon atoms, whereinthe alkyl chain is optionally interrupted by one or more ester bonds;and

z has mean value ranging from 30 to 60.

In some of the foregoing embodiments of the pegylated lipid (II), R¹⁰and R¹¹ are not both n-octadecyl when z is 42. In some embodiments, R¹⁰and R¹¹ are each independently a straight or branched, saturated orunsaturated alkyl chain containing from 10 to 18 carbon atoms. In someembodiments, R¹⁰ and R¹¹ are each independently a straight or branched,saturated or unsaturated alkyl chain containing from 12 to 16 carbonatoms. In some embodiments, R¹⁰ and R¹¹ are each independently astraight or branched, saturated or unsaturated alkyl chain containing 12carbon atoms. In some embodiments, R¹⁰ and R¹¹ are each independently astraight or branched, saturated or unsaturated alkyl chain containing 14carbon atoms. In other embodiments, R¹⁰ and R¹¹ are each independently astraight or branched, saturated or unsaturated alkyl chain containing 16carbon atoms. In still more embodiments, R¹⁰ and R¹¹ are eachindependently a straight or branched, saturated or unsaturated alkylchain containing 18 carbon atoms. In still other embodiments, R¹⁰ is astraight or branched, saturated or unsaturated alkyl chain containing 12carbon atoms and R¹¹ is a straight or branched, saturated or unsaturatedalkyl chain containing 14 carbon atoms.

In various embodiments, z spans a range that is selected such that thePEG portion of (II) has an average molecular weight of about 400 toabout 6000 g/mol. In some embodiments, the average z is about 45.

In other embodiments, the pegylated lipid has one of the followingstructures:

wherein n spans a range such that the average molecular weight of thepegylated lipid is about 2500 g/mol.

Compositions comprising (II) and a cationic lipid are also provided. Thecationic lipid may be selected from any cationic lipid. In variousembodiments, the cationic lipid is a compound having structure (I) asdescribed above, including any of the substructures and specificcompounds in Table 1.

In some embodiments, compositions comprising any one or more of thecompounds of Formula (I) are provided. For example, in some embodiments,the compositions comprise any of the compounds of Formula (I) and atherapeutic agent and one or more excipient selected from neutrallipids, steroids and pegylated lipids. Other pharmaceutically acceptableexcipients and/or carriers are also included in various embodiments ofthe compositions.

In certain embodiments, the therapeutic agent comprises a nucleic acid,for example an antisense oligonucleotide or messenger RNA. In someembodiments, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC,POPC, DOPE and SM. In various embodiments, the molar ratio of thecompound to the neutral lipid ranges from about 2:1 to about 8:1.

In various embodiments, the compositions further comprise a steroid orsteroid analogue. In certain embodiments, the steroid or steroidanalogue is cholesterol. In some of these embodiments, the molar ratioof the compound to cholesterol ranges from about 2:1 to 1:1.

In various embodiments, the composition comprises a pegylated lipid. Forexample, some embodiments include PEG-DMG. In various embodiments, themolar ratio of the compound to the pegylated lipid ranges from about100:1 to about 25:1.

In some embodiments, the composition comprises a pegylated lipid havingthe following structure (II):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein:

R¹⁰ and R¹¹ are each independently a straight or branched, saturated orunsaturated alkyl chain containing from 10 to 30 carbon atoms, whereinthe alkyl chain is optionally interrupted by one or more ester bonds;and

z has a mean value ranging from 30 to 60.

In some embodiments, R¹⁰ and R¹¹ are each independently straight,saturated alkyl chains containing from 12 to 16 carbon atoms. In otherembodiments, the average z is about 45.

In some embodiments of the foregoing composition, the therapeutic agentcomprises a nucleic acid. For example, in some embodiments, the nucleicacid is selected from antisense, plasmid DNA and messenger RNA.

For the purposes of administration, the compounds of the presentinvention (typically in the form of lipid nanoparticles in combinationwith a therapeutic agent) may be administered as a raw chemical or maybe formulated as pharmaceutical compositions. Pharmaceuticalcompositions of the present invention comprise a compound of Formula (I)and one or more pharmaceutically acceptable carrier, diluent orexcipient. The compound of Formula (I) is present in the composition inan amount which is effective to form a lipid nanoparticle and deliverthe therapeutic agent, e.g., for treating a particular disease orcondition of interest. Appropriate concentrations and dosages can bereadily determined by one skilled in the art.

Administration of the compositions of the invention can be carried outvia any of the accepted modes of administration of agents for servingsimilar utilities. The pharmaceutical compositions of the invention maybe formulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suspensions, suppositories, injections, inhalants, gels,microspheres, and aerosols. Typical routes of administering suchpharmaceutical compositions include, without limitation, oral, topical,transdermal, inhalation, parenteral, sublingual, buccal, rectal,vaginal, and intranasal. The term parenteral as used herein includessubcutaneous injections, intravenous, intramuscular, intradermal,intrasternal injection or infusion techniques. Pharmaceuticalcompositions of the invention are formulated so as to allow the activeingredients contained therein to be bioavailable upon administration ofthe composition to a patient. Compositions that will be administered toa subject or patient take the form of one or more dosage units, wherefor example, a tablet may be a single dosage unit, and a container of acompound of the invention in aerosol form may hold a plurality of dosageunits. Actual methods of preparing such dosage forms are known, or willbe apparent, to those skilled in this art; for example, see Remington:The Science and Practice of Pharmacy, 20th Edition (Philadelphia Collegeof Pharmacy and Science, 2000). The composition to be administered will,in any event, contain a therapeutically effective amount of a compoundof the invention, or a pharmaceutically acceptable salt thereof, fortreatment of a disease or condition of interest in accordance with theteachings of this invention.

A pharmaceutical composition of the invention may be in the form of asolid or liquid. In one aspect, the carrier(s) are particulate, so thatthe compositions are, for example, in tablet or powder form. Thecarrier(s) may be liquid, with the compositions being, for example, anoral syrup, injectable liquid or an aerosol, which is useful in, forexample, inhalatory administration.

When intended for oral administration, the pharmaceutical composition ispreferably in either solid or liquid form, where semi-solid,semi-liquid, suspension and gel forms are included within the formsconsidered herein as either solid or liquid.

As a solid composition for oral administration, the pharmaceuticalcomposition may be formulated into a powder, granule, compressed tablet,pill, capsule, chewing gum, wafer or the like form. Such a solidcomposition will typically contain one or more inert diluents or ediblecarriers. In addition, one or more of the following may be present:binders such as carboxymethylcellulose, ethyl cellulose,microcrystalline cellulose, gum tragacanth or gelatin; excipients suchas starch, lactose or dextrins, disintegrating agents such as alginicacid, sodium alginate, Primogel, corn starch and the like; lubricantssuch as magnesium stearate or Sterotex; glidants such as colloidalsilicon dioxide; sweetening agents such as sucrose or saccharin; aflavoring agent such as peppermint, methyl salicylate or orangeflavoring; and a coloring agent.

When the pharmaceutical composition is in the form of a capsule, forexample, a gelatin capsule, it may contain, in addition to materials ofthe above type, a liquid carrier such as polyethylene glycol or oil.

The pharmaceutical composition may be in the form of a liquid, forexample, an elixir, syrup, solution, emulsion or suspension. The liquidmay be for oral administration or for delivery by injection, as twoexamples. When intended for oral administration, preferred compositioncontain, in addition to the present compounds, one or more of asweetening agent, preservatives, dye/colorant and flavor enhancer. In acomposition intended to be administered by injection, one or more of asurfactant, preservative, wetting agent, dispersing agent, suspendingagent, buffer, stabilizer and isotonic agent may be included.

The liquid pharmaceutical compositions of the invention, whether they besolutions, suspensions or other like form, may include one or more ofthe following adjuvants: sterile diluents such as water for injection,saline solution, preferably physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono ordiglycerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose; agents to act as cryoprotectants such assucrose or trehalose. The parenteral preparation can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile.

A liquid pharmaceutical composition of the invention intended for eitherparenteral or oral administration should contain an amount of a compoundof the invention such that a suitable dosage will be obtained.

The pharmaceutical composition of the invention may be intended fortopical administration, in which case the carrier may suitably comprisea solution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, bee wax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device.

The pharmaceutical composition of the invention may be intended forrectal administration, in the form, for example, of a suppository, whichwill melt in the rectum and release the drug. The composition for rectaladministration may contain an oleaginous base as a suitablenonirritating excipient. Such bases include, without limitation,lanolin, cocoa butter and polyethylene glycol.

The pharmaceutical composition of the invention may include variousmaterials, which modify the physical form of a solid or liquid dosageunit. For example, the composition may include materials that form acoating shell around the active ingredients. The materials that form thecoating shell are typically inert, and may be selected from, forexample, sugar, shellac, and other enteric coating agents.Alternatively, the active ingredients may be encased in a gelatincapsule.

The pharmaceutical composition of the invention in solid or liquid formmay include an agent that binds to the compound of the invention andthereby assists in the delivery of the compound. Suitable agents thatmay act in this capacity include a monoclonal or polyclonal antibody, ora protein.

The pharmaceutical composition of the invention may consist of dosageunits that can be administered as an aerosol. The term aerosol is usedto denote a variety of systems ranging from those of colloidal nature tosystems consisting of pressurized packages. Delivery may be by aliquefied or compressed gas or by a suitable pump system that dispensesthe active ingredients. Aerosols of compounds of the invention may bedelivered in single phase, bi-phasic, or tri-phasic systems in order todeliver the active ingredient(s). Delivery of the aerosol includes thenecessary container, activators, valves, subcontainers, and the like,which together may form a kit. One skilled in the art, without undueexperimentation may determine preferred aerosols.

The pharmaceutical compositions of the invention may be prepared bymethodology well known in the pharmaceutical art. For example, apharmaceutical composition intended to be administered by injection canbe prepared by combining the lipid nanoparticles of the invention withsterile, distilled water or other carrier so as to form a solution. Asurfactant may be added to facilitate the formation of a homogeneoussolution or suspension. Surfactants are compounds that non-covalentlyinteract with the compound of the invention so as to facilitatedissolution or homogeneous suspension of the compound in the aqueousdelivery system.

The compositions of the invention, or their pharmaceutically acceptablesalts, are administered in a therapeutically effective amount, whichwill vary depending upon a variety of factors including the activity ofthe specific therapeutic agent employed; the metabolic stability andlength of action of the therapeutic agent; the age, body weight, generalhealth, sex, and diet of the patient; the mode and time ofadministration; the rate of excretion; the drug combination; theseverity of the particular disorder or condition; and the subjectundergoing therapy.

Compositions of the invention may also be administered simultaneouslywith, prior to, or after administration of one or more other therapeuticagents. Such combination therapy includes administration of a singlepharmaceutical dosage formulation of a composition of the invention andone or more additional active agents, as well as administration of thecomposition of the invention and each active agent in its own separatepharmaceutical dosage formulation. For example, a composition of theinvention and the other active agent can be administered to the patienttogether in a single oral dosage composition such as a tablet orcapsule, or each agent administered in separate oral dosageformulations. Where separate dosage formulations are used, the compoundsof the invention and one or more additional active agents can beadministered at essentially the same time, i.e., concurrently, or atseparately staggered times, i.e., sequentially; combination therapy isunderstood to include all these regimens.

Preparation methods for the above compounds and compositions aredescribed herein below and/or known in the art.

It will be appreciated by those skilled in the art that in the processdescribed herein the functional groups of intermediate compounds mayneed to be protected by suitable protecting groups. Such functionalgroups include hydroxy, amino, mercapto and carboxylic acid. Suitableprotecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl(for example, t-butyldimethylsilyl, t-butyldiphenylsilyl ortrimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitableprotecting groups for amino, amidino and guanidino includet-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protectinggroups for mercapto include —C(O)—R″ (where R″ is alkyl, aryl orarylalkyl), p-methoxybenzyl, trityl and the like. Suitable protectinggroups for carboxylic acid include alkyl, aryl or arylalkyl esters.Protecting groups may be added or removed in accordance with standardtechniques, which are known to one skilled in the art and as describedherein. The use of protecting groups is described in detail in Green, T.W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rdEd., Wiley. As one of skill in the art would appreciate, the protectinggroup may also be a polymer resin such as a Wang resin, Rink resin or a2-chlorotrityl-chloride resin.

It will also be appreciated by those skilled in the art, although suchprotected derivatives of compounds of this invention may not possesspharmacological activity as such, they may be administered to a mammaland thereafter metabolized in the body to form compounds of theinvention which are pharmacologically active. Such derivatives maytherefore be described as “prodrugs”. All prodrugs of compounds of thisinvention are included within the scope of the invention.

Furthermore, all compounds of the invention which exist in free base oracid form can be converted to their pharmaceutically acceptable salts bytreatment with the appropriate inorganic or organic base or acid bymethods known to one skilled in the art. Salts of the compounds of theinvention can be converted to their free base or acid form by standardtechniques.

The following Reaction Scheme illustrates methods to make compounds ofthis invention, i.e., compounds of formula (I):

wherein R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R^(4a), R^(4b),R⁵, R⁶, R⁷, R⁸, R⁹, a, b, c, d and e are as defined herein. It isunderstood that one skilled in the art may be able to make thesecompounds by similar methods or by combining other methods known to oneskilled in the art. It is also understood that one skilled in the artwould be able to make, in a similar manner as described below, othercompounds of Formula (I) not specifically illustrated below by using theappropriate starting components and modifying the parameters of thesynthesis as needed. In general, starting components may be obtainedfrom sources such as Sigma Aldrich, Lancaster Synthesis, Inc.,Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc. orsynthesized according to sources known to those skilled in the art (see,for example, Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, 5th edition (Wiley, December 2000)) or prepared as describedin this invention.

Embodiments of the compound of structure (I) (e.g., compound A-5) can beprepared according to General Reaction Scheme 1 (“Method A”), wherein Ris a saturated or unsaturated C₁-C₂₄ alkyl or saturated or unsaturatedcycloalkyl, m is 0 or 1 and n is an integer from 1 to 24. Referring toGeneral Reaction Scheme 1, compounds of structure A-1 can be purchasedfrom commercial sources or prepared according to methods familiar to oneof ordinary skill in the art. A mixture of A-1, A-2 and DMAP is treatedwith DCC to give the bromide A-3. A mixture of the bromide A-3, a base(e.g., N,N-diisopropylethylamine) and the N,N-dimethyldiamine A-4 isheated at a temperature and time sufficient to produce A-5 after anynecessarily workup and or purification step.

Embodiments of the compound of structure (I) (e.g., compound B-5) can beprepared according to General Reaction Scheme 2 (“Method B”), wherein Ris a saturated or unsaturated C₁-C₂₄ alkyl or saturated or unsaturatedcycloalkyl, m is 0 or 1 and n is an integer from 1 to 24. As shown inGeneral Reaction Scheme 2, compounds of structure B-1 can be purchasedfrom commercial sources or prepared according to methods familiar to oneof ordinary skill in the art. A solution of B-1 (1 equivalent) istreated with acid chloride B-2 (1 equivalent) and a base (e.g.,triethylamine). The crude product is treated with an oxidizing agent(e.g., pyridinum chlorochromate) and intermediate product B-3 isrecovered. A solution of crude B-3, an acid (e.g., acetic acid), andN,N-dimethylaminoamine B-4 is then treated with a reducing agent (e.g.,sodium triacetoxyborohydride) to obtain B-5 after any necessary work upand/or purification.

It should be noted that although starting materials A-1 and B-1 aredepicted above as including only saturated methylene carbons, startingmaterials which include carbon-carbon double bonds may also be employedfor preparation of compounds which include carbon-carbon double bonds.

Embodiments of the compound of structure (I) (e.g., compound C-7 or C9)can be prepared according to General Reaction Scheme 3 (“Method C”),wherein R is a saturated or unsaturated C₁-C₂₄ alkyl or saturated orunsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24.Referring to General Reaction Scheme 3, compounds of structure C-1 canbe purchased from commercial sources or prepared according to methodsfamiliar to one of ordinary skill in the art.

The following examples are provided for purpose of illustration and notlimitation.

Example 1 Synthesis of Compound 1

Compound 1 was prepared according to method B as follows:

A solution of octan-1,8-diol (9.8 g) in methylene chloride (100 mL) andtetrahydrofuran (60 mL) was treated with 2-ethylhexanoyl chloride (10g). Triethylamine (15 mL) was slowly added and the solution stirred forthree days. The reaction mixture was filtered and the filtrate washedwith brine (2×). The organic fraction was dried over anhydrous magnesiumsulfate, filtered and the solvent removed. The crude product wasfiltered through silica gel (20 g) using methylene chloride, yielding15.8 g of crude product. The resultant oil was dissolved in methylenechloride (100 mL) and treated with pyridinum chlorochromate (13 g) fortwo hours. Diethyl ether (400 mL) as added and the supernatant filteredthrough a silica gel bed. The solvent was removed from the filtrate andresultant oil passed down a silica gel (77 g) column using a ethylacetate/hexane (0-6%) gradient. 8-O-(2′-ethylhexanoyloxy)octanal (6.7 g)was recovered as an oil.

A solution of 8-O-(2′-ethylhexanoyloxy)octanal (6.7 g), acetic acid (25drops) and 2-N,N-dimethylaminoethylamine (0.54 g) in methylene chloride(40 mL) was treated with sodium triacetoxyborohydride (1.5 g) overnight.The solution was washed with aqueous sodium hydrogen carbonate, followedby brine. The organic phase was dried over anhydrous magnesium sulfate,filtered and the solvent removed. The residue was passed down a silicagel (75 g) column using a methanol/methylene chloride (0-10%) gradient,followed by a second column (20 g), to yield compound 1 (1 g) as acolorless oil.

Example 2 Synthesis of Compound 2

Compound 2 was prepared according to method A as follows:

Under an argon atmosphere, to a round-bottom flask charged with phytol(593 mg, 2 mmol), 6-bromohexanoic acid (780 mg, 4 mmol) and4-(dimethylamino)pyridine (60 mg) in dichloromethane (20 mL) was addeddicyclohexylcarbodiimide (908 mg, 4.4 mmol). The precipitate wasdiscarded by filtration. The filtrate was concentrated and the resultingresidue was purified by column chromatography on silica gel eluted witha gradient mixture (0% to 3%) of ethyl acetate in hexanes. This gave acolorless oil (0.79 g 1.67 mmol, 83%) of(E)-3,7,11,15-tetramethylhexadec-2-enyl 6-bromohexanoate.

A solution of (E)-3,7,11,15-tetramethylhexadec-2-enyl 6-bromohexanoate(0.42 g, 0.887 mmol), N,N-diisopropylethylamine (1.5 mol eq., 1.33 mmol,MW 129.25, 171 mg) and N,N-dimethylethylenediamine (39 mg, 0.44 mmol) inDMF (4 mL) was heated at 77 C for 18 h. The reaction mixture was thencooled and extracted with hexanes (3×20 mL). The hexane extracts werecombined, dried over sodium sulfate, filtered and concentrated. This iscombined with 2^(nd) reaction (total about 0.7 g). The crude waspurified several times by column chromatography on silica gel elutedwith a gradient mixture (0% to 5%) of methanol in DCM. This gave aslightly yellow oil (39 mg) of the desired product. ¹HNMR (400 MHz,CDCl₃) δ: 5.33 (m, 2H), 4.59 (m, 4H), 2.85-2.25 (m, 18H).

Example 3 Synthesis of Compound 3

Compound 3 was prepared in a manner analogous to compound 2 startingfrom bromoacetic acid, rather than 6-bromohexanoic acid, to yield 22 mgof thick colorless oil, 0.029 mmol, 6%. 1HNMR (400 MHz, CDCl3) δ: 5.32(m, 2H), 4.62 (m, 4H), 3.62 (s, 2H), 3.60 (s, 2H), 3.28-2.33 (m, 10H),2.09-2.00 (m, 4H), 1.76 (s, 3H), 1.70 (s, 3H), 1.60-1.47 (m, 6H),1.47-0.97 (32H), 0.89-0.84 (m, 24H).

Example 4 Synthesis of Compound 4

Compound 4 was prepared according to method B as follows:

A solution of dodecan-1,12-diol (10 g) in methylene chloride (100 mL)and tetrahydrofuran (50 mL) was treated with 2-ethylhexanoic acid (7.2g), DCC (10.5 g), DMAP (3.5 g) and triethylamine (10 mL). The solutionwas stirred for four days. The reaction mixture was filtered and thefiltrate washed with dilute hydrochloric acid. The organic fraction wasdried over anhydrous magnesium sulfate, filtered and the solventremoved. The residue was dissolved in methylene chloride (50 mL),allowed to stand overnight, and filtered. The solvent was removed toyield 12.1 g crude product.

The crude product dissolved in methylene chloride (100 mL) and treatedwith pyridinum chlorochromate (8 g) overnight. Diethyl ether (400 mL) asadded and the supernatant filtered through a silica gel bed. The solventwas removed from the filtrate and resultant oil passed down a silica gel(75 g) column using a ethyl acetate/hexane (0-6%) gradient. Crude12-O-(2′-ethylhexanoyloxy)dodecanal (3.5 g) was recovered as an oil.

A solution of the crude product (3.5 g), acetic acid (60 drops) and2-N,N-dimethylaminoethylamine (0.30 g) in methylene chloride (20 mL) wastreated with sodium triacetoxyborohydride (0.86 g) overnight. Thesolution was washed with aqueous sodium hydrogen carbonate, followed bybrine. The organic phase was dried over anhydrous magnesium sulfate,filtered and the solvent removed. The residue was passed down a silicagel (20 g) column using a methanol/methylene chloride (0-8%) gradient,followed by a second column (20 g), to yield the desired product as a(0.6 g) as a colorless oil.

Example 5 Synthesis of Compound 5

Compound 5 was prepared according to method B as follows:

A solution of hexan-1,6-diol (10 g) in methylene chloride (40 mL) andtetrahydrofuran (20 mL) was treated with 2-hexyldecanoyl chloride (10 g)and triethylamine (10 mL). The solution was stirred for an hour and thesolvent removed. The reaction mixture was suspended in hexane, filteredand the filtrate washed with water. The solvent was removed and theresidue passed down a silica gel (50 g) column using hexane, followed bymethylene chloride, as the eluent, yielding6-(2′-hexyldecanoyloxy)hexan-1-ol as an oil (7.4 g).

The purified product (7.4 g) was dissolved in methylene chloride (50 mL)and treated with pyridinum chlorochromate (5.2 g) for two hours. Diethylether (200 mL) as added and the supernatant filtered through a silicagel bed. The solvent was removed from the filtrate and resultant oilpassed down a silica gel (50 g) column using a ethyl acetate/hexane(0-5%) gradient. 6-(2′-hexyldecanoyloxy)dodecanal (5.4 g) was recoveredas an oil.

A solution of the product (4.9 g), acetic acid (0.33 g) and2-N,N-dimethylaminoethylamine (0.40 g) in methylene chloride (20 mL) wastreated with sodium triacetoxyborohydride (2.1 g) for two hours. Thesolution was washed with aqueous sodium hydroxide. The organic phase wasdried over anhydrous magnesium sulfate, filtered and the solventremoved. The residue was passed down a silica gel (50 g) column using amethanol/methylene chloride (0-8%) gradient to yield the desired product(1.4 g) as a colorless oil.

Example 6 Synthesis of Compound 6

Compound 6 was prepared according to method B as follows:

A solution of nonan-1,9-diol (12.6 g) in methylene chloride (80 mL) wastreated with 2-hexyldecanoic acid (10.0 g), DCC (8.7 g) and DMAP (5.7g). The solution was stirred for two hours. The reaction mixture wasfiltered and the solvent removed. The residue was dissolved in warmedhexane (250 mL) and allowed to crystallize. The solution was filteredand the solvent removed. The residue was dissolved in methylene chlorideand washed with dilute hydrochloric acid. The organic fraction was driedover anhydrous magnesium sulfate, filtered and the solvent removed. Theresidue was passed down a silica gel column (75 g) using 0-12% ethylacetate/hexane as the eluent, yielding 9-(2′-hexyldecanoyloxy)nonan-1-ol(9.5 g) as an oil.

The product was dissolved in methylene chloride (60 mL) and treated withpyridinum chlorochromate (6.4 g) for two hours. Diethyl ether (200 mL)as added and the supernatant filtered through a silica gel bed. Thesolvent was removed from the filtrate and resultant oil passed down asilica gel (75 g) column using a ethyl acetate/hexane (0-12%) gradient,yielding 9-(2′-ethylhexanoyloxy)nonanal (6.1 g) as an oil.

A solution of the crude product (6.1 g), acetic acid (0.34 g) and2-N,N-dimethylaminoethylamine (0.46 g) in methylene chloride (20 mL) wastreated with sodium triacetoxyborohydride (2.9 g) for two hours. Thesolution was diluted with methylene chloride washed with aqueous sodiumhydroxide, followed by water. The organic phase was dried over anhydrousmagnesium sulfate, filtered and the solvent removed. The residue waspassed down a silica gel (75 g) column using a methanol/methylenechloride (0-8%) gradient, followed by a second column (20 g) using amethylene chloride/acetic acid/methanol gradient. The purified fractionswere dissolved in methylene chloride, washed with dilute aqueous sodiumhydroxide solution, dried over anhydrous magnesium sulfate, filtered andthe solvent removed, to yield the desired product (1.6 g) as a colorlessoil.

Example 7 Synthesis of Compound 7

Compound 7 was prepared from 3,5,5-trimethylhexyl 10-bromodecanoate andN,N-dimethylethane-1,2-diamine according to method A to yield 144 mg ofslightly yellow oil, 0.21 mmol, 11%). 1HNMR (400 MHz, CDCl3) δ: 4.09(t-like, 6.6 Hz, 4H), 2.58-2.51 (m, 2H), 2.44-2.34 (m, 6H), 2.29(t-like, 7.5 Hz, 4H), 2.25 (s, 6H), 1.67-1.57 (m, 8H), 1.52-1.39 (m,6H), 1.36-1.21 (m, 24H), 0.95 (d, 6.6 Hz, 6H), 0.90 (s, 18H).

Example 8 Synthesis of Compound 8

Compound 8 was prepared by method A in 15% yield. ¹HNMR (400 MHz, CDCl3)δ: 5.11-5.04 (m, 2H), 2.60-2.54 (m, 2H), 2.47-2.36 (m, 6H), 2.27(t-like, 7.4 Hz, 4H), 2.25 (s, 6H), 1.66-1.40 (m, 16H), 1.34-1.23 (m,24H), 0.91 (d, 6.5 Hz, 24H).

Example 9 Synthesis of Compound 9

Compound 9 was prepared according to method B as follows:

A solution of nonan-1,9-diol (10.0 g) in methylene chloride (100 mL) wastreated with citroneloyl chloride (10.1 g, prepared from citronelic acidand oxalyl chloride) and triethylamine (10 mL), and stirred for threedays. The reaction mixture was diluted with methylene chloride andwashed with dilute hydrochloric acid. The organic fraction was driedover anhydrous magnesium sulfate, filtered and the solvent removed. Theresidue was taken up in hexane, filtered and the solvent removed. Theresidue was passed down a series of silica gel columns (60-70 g) usinghexane followed by methylene chloride as the eluent, yielding9-(citroneloyloxy)nonan-1-ol (7.6 g) as an oil.

The product was dissolved in methylene chloride (50 mL) and treated withpyridinum chlorochromate (6.4 g) for 90 minutes. Diethyl ether (200 mL)as added and the supernatant filtered through a silica gel bed. Theresidue was dissolved in hexane and passed through a silica gel (20 g)column using hexane as the eluent, yielding 9-(citroneloyloxy)nonanal (5g) as an oil.

A solution of the crude product (5 g), acetic acid (0.33 g) and2-N,N-dimethylaminoethylamine (0.48 g) in methylene chloride (40 mL) wastreated with sodium triacetoxyborohydride (1.2 g) overnight. Thesolution was diluted with methylene chloride and washed with aqueoussodium hydroxide. The organic phase was dried over anhydrous magnesiumsulfate, filtered and the solvent removed. The residue was passed down asilica gel (50 g) column using a 0-12% methanol/methylene chloridegradient, followed by a second silica gel column (20 g) using the samegradient, to yield the desired product (0.6 g) as a colorless oil.

Example 10 Synthesis of Compound 10

Compound 10 was prepared according to method A to yield 147 mg ofcolorless oil, 0.23 mmol, 17%. ¹HNMR (400 MHz, CDCl3) δ: 4.11 (t, 6.9Hz, 4H), 2.56-2.52 (m, 2H), 2.44-2.35 (m, 6H), 2.29 (t-like, 7.5 Hz,4H), 2.24 (s, 6H), 1.75-1.66 (m, 8H), 1.66-1.57 (m, 4H), 1.52 (q-like,6.9 Hz, 4H), 1.46-1.38 (m, 4H), 1.38-1.13 (m, 30H), 0.98-0.87 (m, 4H).

Example 11 Synthesis of Compound 11

Compound 11 was prepared according to method A to yield 154 mg ofslightly yellow oil, 0.22 mmol, 14%). ¹HNMR (400 MHz, CDCl3) δ: 4.88(quintet, 6.2 Hz, 2H), 3.20-2.40 (m, 8H), 2.39 (s, 6H), 2.29 (t, 7.5 Hz,4H), 1.67-1.56 (m, 4H), 1.56-1.48 (m, 8H), 1.38-1.21 (m, 44H), 0.92-0.86(m, 12H).

Example 12 Synthesis of Compound 12

Compound 12 was prepared according to method A to yield 169 mg ofslightly yellow oil, 0.26 mmol, 17%). ¹HNMR (400 MHz, CDCl3) δ:4.03-3.95 (ABX pattern, 4H), 2.54 (m, 2H), 2.44-2.35 (m, 6H), 2.30(t-like, 7.5 Hz, 4H), 2.25 (s, 6H), 1.66-1.54 (m, 6H), 1.47-1.23 (m,40H), 0.92-0.88 (m, 12H).

Example 13 Synthesis of Compound 13

Compound 13 was prepared according to method A to yield 152 mg of whitepaste, 0.23 mmol, 16%. ¹HNMR (400 MHz, CDCl3) δ: 4.03 (t, 6.7 Hz, 4H),3.10-2.41 (very broad, 8H), 2.34 (s, 6H), 2.30 (t, 7.5 Hz, 4H),1.66-1.46 (m, 12H), 1.39-1.21 (m, 40H), 0.89 (t-like, 6.9 Hz, 6H).

Example 14 Synthesis of Compound 14

Compound 14 was prepared according to method A to yield 111 mg ofcolorless oil, 0.16 mmol, 11%. ¹HNMR (400 MHz, CDCl3) δ: 5.09 (m, 2H),4.16-4.05 (m, 4H), 3.10-2.40 (very broad, 8H), 2.31 (s, 6H), 2.29 (t,7.5 Hz, 4H), 2.06-1.89 (m, 4H), 1.69 (d, 0.8 Hz, 6H), 1.61 (s, 6H),1.73-1.13 (m, 50H), 0.92 (d, 6.6 Hz, 6H).

Example 15 Synthesis of Compound 15

Compound 15 was prepared according to method A to yield 116 mg of whitepaste, 0.16 mmol, 10%. ¹HNMR (400 MHz, CDCl3) δ: 4.06 (t, 6.7 Hz, 4H),2.62-2.51 (broad, 2H), 2.48-2.33 (br., 6H), 2.29 (t, 7.5 Hz, 4H), 2.25(s, 6H), 1.69 (quintet, 7.0 Hz, 8H), 1.48-1.38 (br., 4H), 1.38-1.21 (m,52H), 0.89 (t-like, 6.8 Hz, 6H).

Example 16 Synthesis of Compound 16

Compound 16 was prepared according to method A to yield 118 mg ofcolorless oil 0.17 mmol, 12%. ¹HNMR (400 MHz, CDCl3) δ: 4.06 (t, 6.8 Hz,4H), 2.57-2.52 (m, 2H), 2.44-2.34 (m, 6H), 2.29 (t, 7.6 Hz, 4H), 2.25(s, 6H), 1.62 (quintet-like, 7.0 Hz, 8H), 1.47-1.39 (m, 4H), 1.37-1.22(m, 44H), 0.89 (t-like, 6.8 Hz, 6H).

Example 17 Synthesis of Compound 17

Compound 17 was prepared according to method A to yield 145 mg ofslightly yellow oil, 0.21 mmol, 13%. 1HNMR (400 MHz, CDCl3) δ: 5.01 (m,0.27H from cis-isomer), 4.63 (tt, 11.2 Hz, 4.5 Hz, 1.73H fromtrans-isomer), 2.61-2.24 (18H), 2.01 (m, 4H), 1.81 (m, 4H), 1.61(quintet-like, 7.2 Hz, 4H), 1.44 (m, 4H), 1.36-1.21 (24H), 1.11 (m, 4H),1.01 (m, 2H), 0.87 (s, 2.7H from cis-isomer), 0.86 (s, 15.3H fromtrans-isomer).

Example 18 Synthesis of Compound 18

Compound 18 was prepared according to method A to yield 111 mg ofcolorless oil, 0.17 mmol, 14%. ¹HNMR (400 MHz, CDCl3) δ: 4.88 (quintet,6.2 Hz, 2H), 2.61-2.51 (br., 2H), 2.48-2.34 (br, 6H), 2.29 (t, 7.6 Hz,4H), 2.25 (s, 6H), 1.62 (quintet-like, 7.3 Hz, 4H), 1.55-1.48 (m, 8H),1.47-1.39 (m, 4H), 1.37-1.21 (m, 32H), 0.91-0.86 (m, 12H).

Example 19 Synthesis of Compound 19

Compound 19 was prepared according to method A to yield 76 mg ofcolorless oil, 0.11 mmol, 6%. ¹HNMR (400 MHz, CDCl3) δ: 5.77 (dt-like,14.4 Hz, 6.6 Hz, 2H), 5.55 (dtt-like, 14.4 Hz, 6.5 Hz, 1.4 Hz, 2H), 4.51(dd, 6.6 Hz, 0.6 Hz, 4H), 2.61-2.50 (br., 2H), 2.50-2.34 (br. 6H), 2.30(t, 7.5 Hz, 4H), 2.25 (s, 6H), 2.04 (q, 7.1 Hz, 4H), 1.62 (quintet, 7.3Hz, 4H), 1.48-1.21 (40H), 0.88 (t-like, 6.8 Hz, 6H).

Example 20 Synthesis of Compound 20

Compound 20 was prepared according to the general procedure A to yield157 mg of colorless oil, 0.22 mmol, 14%. ¹HNMR (400 MHz, CDCl3) δ: 3.97(d, 5.8 Hz, 4H), 2.57-2.51 (m, 2H), 2.44-2.33 (m, 6H), 2.30 (t, 7.5 Hz,4H), 2.24 (s, 6H), 1.63 (quintet-like, 7.3 Hz, 6H), 1.43 (quintet-like,7.3 Hz, 4H), 1.36-1.21 (44H), 0.93-0.86 (m, 12H).

Example 21 Synthesis of Compound 21

Compound 21 was prepared according to the general procedure A to yield164 mg of colorless oil, 0.21 mmol, 14%. ¹HNMR (400 MHz, CDCl3) δ: 3.97(d, 5.8 Hz, 4H), 2.57-2.51 (m, 2H), 2.44-2.34 (m, 6H), 2.30 (t, 7.5 Hz,4H), 2.24 (s, 6H), 1.62 (quintet-like, 7.3 Hz, 6H), 1.43 (quintet-like,7.3 Hz, 4H), 1.36-1.21 (52H), 0.93-0.86 (m, 12H).

Example 22 Synthesis of Compound 22

Compound 22 was prepared according to method A as follows:

Step 1.

To a solution of 6-bromohexanoic acid (20 mmol, 3.901 g),2-hexyl-1-decanol (1.8 eq, 36 mmol, 8.72 g) and 4-dimethylaminopyridine(DMAP 0.5 eq, 10 mmol, 1.22 g) in DCM (80 mL) was added DCC (1.1 eq, 22mmol, 4.54 g). The resulting mixture was stirred at RT for 16 h. Theprecipitate was discarded by filtration. The filtrate was concentrated.The residue was purified by column chromatography on silica gel elutedwith a gradient mixture of ethyl acetate in hexanes (0 to 2%). This gavethe desired product as a colorless oil (7.88 g, 18.8 mmol, 94%)

Step 2.

A mixture of the bromide from step 1 (1.34 equiv., 7.88 g, 18.8 mmol),N,N-diisopropylethylamine (1.96 equiv., 27.48 mmol, 4.78 mL) andN,N-dimethylethylenediamine (1 equiv., 14.02 mmol, 1.236 g, 1.531 mL) inacetonitrile (70 mL) in 250 mL flask equipped with a condenser washeated at 79 C (oil bath) for 16 h. The reaction mixture was cooled toRT and concentrate. The residue was taken in a mixture of ethyl acetateand hexanes (1:9) and water. The phases were separated, washed withwater (100 mL) and brine. Dried over sodium sulfate and concentrated(8.7 g oil). The crude (8.7 Vg oil) was purified by columnchromatography on silica gel (0 to 3% MeOH in chloroform). The fractionscontaining the desired product were combined and concentrated. Theresidue was dissolved in 1 mL of hexane and filtered through a layer ofsilica gel (3-4 mm, washed with 8 mL of hexane). The filtrate was blownto dry with a stream of Ar and dried well in vacuo overnight (1.30 g,mmol, %, colorless oil, desired product). ¹HNMR (400 MHz, CDCl3) δ: 3.96(d, 5.8 Hz, 4H), 2.55-2.50 (m, 2H), 2.43-2.39 (m, 4H), 3.37-3.32 (m,2H), 2.30 (t, 7.5 Hz, 4H), 2.23 (s, 6H), 1.63 (quintet-like, 7.6 Hz,6H), 1.48-1.40 (m, 4H), 1.34-1.20 (52H), 0.88 (t-like, 6.8 Hz, 12H).

Example 23 Synthesis of Compound 23

Compound 23 was prepared according to the general procedure A to yield200 mg of colorless oil, 0.24 mmol, 16%. ¹HNMR (400 MHz, CDCl3) δ: 3.97(d, 5.8 Hz, 4H), 2.57-2.51 (m, 2H), 2.44-2.34 (m, 6H), 2.30 (t, 7.5 Hz,4H), 2.24 (s, 6H), 1.67-1.58 (m, 6H), 1.43 (quintet-like, 7.3 Hz, 4H),1.36-1.21 (60H), 0.89 (t-like, 6.8 Hz, 12H).

Example 24 Synthesis of Compound 24

Compound 24 was prepared according to the general procedure A to yield138 mg of colorless oil, 0.18 mmol, 12%. ¹HNMR (400 MHz, CDCl3) δ: 4.90(sixlet-liked, 6.3 Hz, 2H), 2.63-2.33 (br. 8H), 2.27 (t, 7.5 Hz, 4H),2.26 (s, 6H), 1.66-1.57 (m, 4H), 1.51-1.39 (m, 6H), 1.35-1.21 (54H),1.20 (d, 6.2 Hz, 6H), 0.89 (t-like, 6.8 Hz, 6H).

Example 25 Synthesis of Compound 25

Compound 25 was prepared according to the general procedure A to yield214 mg of colorless oil, 0.24 mmol, 17%. ¹HNMR (400 MHz, CDCl3) δ: 3.97(d, 5.8 Hz, 4H), 2.58-2.52 (m, 2H), 2.45-2.35 (m, 6H), 2.30 (t, 7.5 Hz,4H), 2.25 (s, 6H), 1.62 (quintet-like, 7.0 Hz, 6H), 1.43 (quintet-like,7.0 Hz, 4H), 1.36-1.21 (68H), 0.89 (t-like, 6.7 Hz, 12H).

Example 26 Synthesis of Compound 26

Compound 26 was prepared according to the general procedure A to yield170 mg of colorless oil, 0.21 mmol, 13%. ¹HNMR (400 MHz, CDCl3) δ:5.42-5.29 (m, 8H), 4.05 (t, 6.8 Hz, 4H), 2.77 (t, 6.5 Hz, 4H), 2.55-2.50(m, 2H), 2.43-2.39 (m, 4H), 2.37-2.32 (m, 2H), 2.29 (t, 7.6 Hz, 4H),2.23 (s, 6H), 2.05 (q, 6.8 Hz, 8H), 1.63 (quintet-like, 7.5 Hz, 8H),1.48-1.40 (m, 4H), 1.39-1.23 (36H), 0.90 (t-like, 6.8 Hz, 6H).

Example 27 Synthesis of Compound 27

Compound 27 was prepared according to the general procedure A to yield255 mg of colorless oil, 0.29 mmol, 18%. ¹HNMR (400 MHz, CDCl3) δ: 3.96(d, 5.8 Hz, 4H), 2.55-2.50 (m, 2H), 2.43-2.39 (m, 4H), 3.37-3.32 (m,2H), 2.30 (t, 7.5 Hz, 4H), 2.23 (s, 6H), 1.63 (quintet-like, 7.6 Hz,6H), 1.48-1.40 (m, 4H), 1.34-1.20 (68H), 0.88 (t-like, 6.8 Hz, 12H).

Example 28 Synthesis of Compound 28

Compound 28 was prepared according to the general procedure A to yield248 mg of colorless oil, 0.27 mmol, 19%. ¹HNMR (400 MHz, CDCl3) δ: 3.97(d, 5.8 Hz, 4H), 2.57-2.52 (m, 2H), 2.44-2.34 (m, 6H), 2.30 (t, 7.5 Hz,4H), 2.24 (s, 6H), 1.67-1.58 (m, 6H), 1.43 (quintet-like, 7.3 Hz, 4H),1.36-1.21 (76H), 0.89 (t-like, 6.8 Hz, 12H).

Example 29 Synthesis of Compound 29

Compound 29 was prepared according to the general procedure A to yield181 mg of colorless oil, 0.23 mmol, 17%. ¹HNMR (400 MHz, CDCl3) δ: 4.87(quintet, 6.3 Hz, 4H), 2.56-2.51 (m, 2H), 2.43-2.34 (m, 6H), 2.27 (t,7.5 Hz, 4H), 2.24 (s, 6H), 1.61 (quintet-like, 7.3 Hz, 4H), 1.55-1.46(m, 8H), 1.46-1.37 (m, 4H), 1.36-1.08 (52H), 0.88 (t-like, 6.8 Hz, 12H).

Example 30 Synthesis of Compound 30

Compound 30 was prepared according to the general procedure A to yield88 mg of colorless oil, 0.11 mmol, 3%. ¹HNMR (400 MHz, CDCl3) δ: 3.97(d, 5.5 Hz, 4H), 2.58-2.51 (m, 2H), 2.49-2.44 (m, 4H), 2.38-2.30 (m,6H), 2.24 (s, 6H), 1.75 (quintet-like, 7.3 Hz, 4H), 1.66-1.54 (m, 2H),1.35-1.06 (64H), 0.89 (t-like, 6.4 Hz, 12H).

Example 31 Synthesis of Compound 31

Compound 31 was prepared according to the general procedure C to yield275 mg of slightly yellow oil, 0.30 mmol, total yield 35% for threesteps. ¹HNMR (400 MHz, CDCl3) δ: 3.97 (d, 5.8 Hz, 4H), 2.63-2.47 (m,8H), 2.45-2.41 (m, 4H), 2.31 (t, 7.5 Hz, 4H), 1.82-1.74 (m, 4H), 1.64(quintet-like, 7.6 Hz, 6H), 1.46 (quintet-like, 7.6 Hz, 4H), 1.36-1.18(68H), 0.89 (t-like, 6.8 Hz, 12H).

Example 32 Synthesis of Compound 32

Compound 32 was prepared according to method C as follows:

Step 1.

To a solution of 2-aminoethanol (116 mg, 1.9 mmol, 115 uL, MW 61.08, d1.012) in 15 ml of anhydrous THF, 2-hexyldecyl 6-bromohexanoate (1.9 eq,1.52 g, 3.62 mmol), potassium carbonate (1.9 eq, 3.62 mmol, 500 mg),cesium carbonate (0.3 eq, 0.57 mmol, 186 mg) and sodium iodide (10 mg)were added and was heated to reflux for 6 days under Ar. The solvent wasevaporated under reduced pressure and the residue was taken up inhexanes and washed with water and brine. The organic layer wasseparated, dried over anhydrous sodium sulphate, filtered and evaporatedunder reduced to obtain a colorless oil. The crude product was purifiedby flash column chromatography on silica gel (230-400 mesh silica gel,MeOH in chloroform, 0 to 4%) to yield 936 mg of colorless oil (1.27mmol, 70%).

Step 2.

To a magnetically stirred and ice-cooled solution of 936 mg (1.27 mmol)of the product from step 1 in 2 mL of CHCl3, was added thionyl chloride(2.9 eq, 3.70 mmol, 440 mg, 270 uL) in 15 mL of chloroform dropwiseunder an Ar atmosphere. After the completion of addition of SOCl2, theice bath was removed and the reaction mixture was stirred for 16 h atroom temperature under an Ar atmosphere. Removal of CHC13, and SOCl2under reduced pressure gave a thick yellow oil.

Step 3.

The crude from step 2 was dissolved in THF (20 mL). To the THF solutionwas added pyrrolidine (1.6 mL, 1.36 g, 19 mmol). The sealed mixture washeated at 64 C overnight. The reaction mixture was concentrated (darkbrown oil). The residue was purified by flash dry column chromatographyon silica gel (MeOH in chloroform, 0 to 4%). This gave the desiredproduct as a slightly yellow oil (419 mg, 0.53 mmol, 83%). ¹HNMR (400MHz, CDCl3) δ: 3.97 (d, 5.8 Hz, 4H), 2.65-2.47 (m, 8H), 2.45-2.41 (m,4H), 2.31 (t, 7.5 Hz, 4H), 1.81-1.74 (m, 4H), 1.64 (quintet-like, 7.6Hz, 6H), 1.46 (quintet-like, 7.6 Hz, 4H), 1.36-1.21 (52H), 0.89 (t-like,6.8 Hz, 12H).

Example 33 Synthesis of Compound 33

Compound 33 was prepared according to the general procedure C to yield419 mg of slightly yellow oil, 0.54 mmol, total yield 60% for threesteps. ¹HNMR (400 MHz, CDCl3) δ: 3.97 (d, 5.8 Hz, 4H), 2.57-2.53 (m,2H), 2.46-2.40 (m, 8H), 2.31 (t, 7.5 Hz, 4H), 2.23 (s, 3H), 1.64(quintet-like, 7.6 Hz, 6H), 1.46 (quintet-like, 7.6 Hz, 4H), 1.36-1.20(52H), 1.06 (t, 7.2 Hz, 3H), 0.89 (t-like, 6.8 Hz, 12H).

Example 34 Synthesis of Compound 34

Compound 34 was prepared according to method B as follows:

A solution of nonan-1,9-diol (10 g) in methylene chloride (250 mL) wastreated with 2-ethylhexanoic acid (4.5 g), DCC (7.7 g) and DMAP (4.2 g).The solution was stirred for three days. The reaction mixture wasfiltered and hexane (200 mL) added to the filtrate. The mixture wasstirred and the precipitates allowed to settle out. The supernatant wasdecanted and the solvent removed. The residue was suspended in hexane(70 mL) and allowed to settle. The supernatant was decanted and thesolvent removed. The residue was dissolved in hexane, allowed to standat room temperature and then filtered. The solvent was removed and theresidue passed down a silica gel column (50 g) using a 0-10% ethylacetate/hexane gradient, followed by a 0-8% methanol/methylene chloridegradient, yielding 5.6 g of 9-(2′ethylhexanoyloxy)nonan-1-ol as acolorless oil.

The product dissolved in methylene chloride (70 mL) and treated withpyridinium chlorochromate (5 g) for two hours. Diethyl ether (250 mL)was added and the supernatant filtered through a silica gel bed. Thesolvent was removed from the filtrate and resultant oil dissolved inhexane. The suspension was filtered through a silica gel plug and thesolvent removed, yielding crude 9-(2′ethylhexanoyloxy)nonanal (3.4 g) asan oil.

A solution of the crude product (3.4 g), acetic acid (0.52 g) and2-N,N-dimethylaminoethylamine (0.33 g) in methylene chloride (50 mL) wastreated with sodium triacetoxyborohydride (1.86 g) overnight. Thesolution was washed with aqueous sodium hydroxide solution. The organicphase was dried over anhydrous magnesium sulfate, filtered and thesolvent removed. The residue was passed down a silica gel (50 g) columnusing an acetic acid/methanol/methylene chloride (2-0%/0-12%/98-88%)gradient. The purified fractions were washed with aqueous sodiumhydrogen carbonate, dried over magnesium sulphate, filtered and thesolvent removed, yielding compound 34 as an oil (0.86 g).

Example 35 Synthesis of Compound 35

Compound 35 was prepared according to method B as follows:

A solution of dodecan-1,12-diol (18.1 g) in methylene chloride (90 mL)was treated with citronelic acid (7.5 g), DCC (10.0 g) and DMAP (9.5 g).The solution was stirred overnight. The reaction mixture was filteredand the filtrate washed with dilute hydrochloric acid. The organicfraction was dried over anhydrous magnesium sulfate, filtered and thesolvent removed to yield 12.2 g of crude 12-citroneloyloxydodecan-1-ol.

The crude product dissolved in methylene chloride (60 mL) and treatedwith pyridinum chlorochromate (6.8 g) for three hours. Diethyl ether(200 mL) as added and the supernatant filtered through a silica gel bed.The solvent was removed from the filtrate and resultant oil passed downa silica gel (75 g) column using a ethyl acetate/hexane (0-12%)gradient. Crude 12-citroneloyloxydodecanal (6.2 g) was recovered as anoil.

A solution of the crude product (6.2 g), acetic acid (0.44 g) and2-N,N-dimethylaminoethylamine (0.50 g) in methylene chloride (40 mL) wastreated with sodium triacetoxyborohydride (2.9 g) overnight. Thesolution was washed with aqueous sodium hydrogen carbonate, followed bybrine. The organic phase was dried over anhydrous magnesium sulfate,filtered and the solvent removed. The residue was passed down a silicagel (75 g) column using an acetic acid/methanol/methylene chloride(2-0%/0-12%/98-88%) gradient. The purified fractions were washed withaqueous sodium hydrogen carbonate, dried over magnesium sulphate,filtered and the solvent removed, yielding compound 35 (1.68 g) as anoil.

Example 36 Synthesis of Compound 36

Compound 36 was prepared according to the general procedure C to yield108 mg of colorless oil (0.14 mmol). ¹HNMR (400 MHz, CDCl3) δ: 4.87(quintet, 6.3 Hz, 2H), 2.56-2.51 (m, 2H), 2.45-2.40 (m, 4H), 2.38-2.33(m, 2H), 2.29 (t, 7.5 Hz, 4H), 2.24 (s, 6H), 1.64 (quintet-like, 7.7 Hz,4H), 1.55-1.41 (m, 12H), 1.35-1.18 (m, 52H), 0.89 (t-like, 6.8 Hz, 12H).

Example 37 Synthesis of Compound 37

Compound 37 was prepared according to the general procedure C to yield330 mg of colorless oil (0.40 mmol, total yield 80% for three steps).¹HNMR (400 MHz, CDCl3) δ: 4.87 (quintet, 6.5 Hz, 2H), 2.64-2.47 (m, 8H),2.45-2.40 (m, 4H), 2.29 (t, 7.5 Hz, 4H), 1.81-1.74 (m, 4H), 1.64(quintet-like, 7.6 Hz, 4H), 1.55-1.41 (m, 12H), 1.35-1.18 (m, 50H), 0.89(t-like, 6.8 Hz, 12H).

Example 38 Synthesis of Compound 38

Compound 38 was prepared according to method B as follows:

A solution of nonan-1,9-diol (16 g) in methylene chloride (100 mL) wastreated with 2-butyloctanoic acid (10 g), DCC (10.3 g) and DMAP (6.7 g).The solution was stirred for three days. The reaction mixture wasfiltered and hexane (250 mL) added to the filtrate. The mixture wasstirred and the precipitates allowed to settle out. The supernatant wasdecanted and the solvent removed. The residue was suspended in hexaneand allowed to settle. The supernatant was decanted and the solventremoved (repeated twice). The residue was dissolved in hexane, allowedto stand at room temperature and then filtered. The solvent was removedand the residue passed down a silica gel column (18 g) using methylenechloride, yielding crude 9-(2′-butyloctanoyloxy)nonan-1-ol (17.7 g) asan oil.

The crude product was dissolved in methylene chloride (250 mL) andtreated with pyridinium chlorochromate (11.2 g) overnight. Diethyl ether(750 mL) was added and the supernatant filtered through a silica gelbed. The solvent was removed from the filtrate and resultant oildissolved in hexane (150 mL). The suspension was filtered through asilica gel plug and the solvent removed. The crude product was passeddown a silica gel (80 g) column using a 0-6% ethyl acetate/hexanegradient, yielding 9-(2′-butyloctanoyloxy)nonanal (5.3 g) as an oil.

A solution of the product (5.3 g), acetic acid (0.37 g) and2-N,N-dimethylaminoethylamine (0.47 g) in methylene chloride (50 mL) wastreated with sodium triacetoxyborohydride (3.35 g) overnight. Thesolution was washed with aqueous sodium hydroxide solution. The organicphase was dried over anhydrous magnesium sulfate, filtered and thesolvent removed. The residue was passed down a silica gel (60 g) columnusing an acetic acid/methanol/methylene chloride (2-0%/0-12%/98-88%)gradient. The purified fractions were washed with aqueous sodiumhydrogen carbonate, dried over magnesium sulphate, filtered and thesolvent removed, yielding compound 38 as an oil (2.3 g).

Example 39 Synthesis of Compound 39

Compound 39 was prepared according to method B as follows:

A solution of hexan-1,6-diol (12 g) in methylene chloride (250 mL) wastreated with 2-decyltetradecanoic acid (17.5 g), DCC (11.3 g) and DMAP(6.8 g). The solution was stirred for overnight. The reaction mixturewas filtered and hexane added to the filtrate. The mixture was stirredand the precipitates allowed to settle out. The supernatant was decantedand the solvent removed. The residue was passed down a silica gel column(80 g) using hexane followed by 0-1% methanol/methylene chloride,yielding crude 6-(2′-decyltetradecanoyloxy)hexan-1-ol (5.8 g) as an oil.

The crude product was dissolved in methylene chloride (70 mL) andtreated with pyridinium chlorochromate (2.9 g) for two hours. Diethylether (250 mL) was added and the supernatant filtered through a silicagel bed. The solvent was removed from the filtrate and resultant oildissolved in hexane. The suspension was filtered through a silica gelplug and the solvent removed. The crude product was passed down a silicagel (10 g) column using a 0-5% ethyl acetate/hexane gradient, yielding6-(2′-decyltetradecanoyloxy)hexanal (3.2 g) as an oil.

A solution of the product (3.2 g), acetic acid (0.28 g) and2-N,N-dimethylaminoethylamine (0.15 g) in methylene chloride (20 mL) wastreated with sodium triacetoxyborohydride (0.98 g) overnight. Thesolution was washed with aqueous sodium hydroxide solution. The organicphase was dried over anhydrous magnesium sulfate, filtered and thesolvent removed. The residue was passed down a silica gel (50 g) columnusing an acetic acid/methanol/methylene chloride (2-0%/0-12%/98-88%)gradient. The purified fractions were washed with aqueous sodiumhydrogen carbonate, dried over magnesium sulphate, filtered and thesolvent removed, yielding compound 39 as an oil (1.2 g).

Example 40 Synthesis of Compound 40

Compound 40 was prepared according to method B as follows:

A solution of nonan-1,9-diol (10.1 g) in methylene chloride (200 mL) wastreated with 2-octyldodecanoic acid (10.0 g), DCC (8.3 g) and DMAP (5.0g). The solution was stirred for overnight. The reaction mixture wasfiltered and hexane (200 mL) added to the filtrate. The mixture wasstirred and the precipitates allowed to settle out. The supernatant wasdecanted and the solvent removed. This process was repeated twice. Theresidue was passed down a silica gel column (75 g) using hexane followedby 4-10% methanol/methylene chloride, yielding crude9-(2′-octyldodecanoyloxy)nonan-1-ol (˜11 g) as an oil.

The crude product was dissolved in methylene chloride (70 mL) andtreated with pyridinium chlorochromate (8 g) for two hours. Diethylether (400 mL) was added and the supernatant filtered through a silicagel bed. The solvent was removed from the filtrate and resultant oildissolved in hexane. The suspension was filtered through a silica gelplug and the solvent removed, yielding crude9-(2′-octyldodecanoyloxy)nonanal (8.4 g) as an oil.

A solution of the product (8.4 g), acetic acid (0.84 g) and2-N,N-dimethylaminoethylamine (0.55 g) in methylene chloride (60 mL) wastreated with sodium triacetoxyborohydride (2.9 g) for two hours. Thesolution was washed with aqueous sodium hydroxide solution. The organicphase was dried over anhydrous magnesium sulfate, filtered and thesolvent removed. The residue was passed down a silica gel (75 g) columnusing an acetic acid/methanol/methylene chloride (2-0%/0-12%/98-88%)gradient. The purified fractions were washed with aqueous sodiumhydrogen carbonate, dried over magnesium sulphate, filtered and thesolvent removed, yielding compound 40 as an oil (3.2 g).

Example 41 Synthesis of Compound 41

Compound 41 was prepared according to method B as follows:

A solution of nonan-1,9-diol (9.6 g) in methylene chloride (200 mL) wastreated with 2-decyltetradecanoic acid (8.4 g), DCC (8.6 g) and DMAP(5.0 g). The solution was stirred for overnight. The reaction mixturewas filtered and hexane (200 mL) added to the filtrate. The mixture wasstirred and the precipitates allowed to settle out. The supernatant wasdecanted and the solvent removed. This process was repeated twice. Theresidue was passed down a silica gel column (75 g) using hexane followedby 4-10% methanol/methylene chloride, yielding crude9-(2′-decyltetradecanoyloxy)nonan-1-ol (6.4 g) as an oil.

The crude product was dissolved in methylene chloride (50 mL) andtreated with pyridinium chlorochromate (5.7 g) for two hours. Diethylether (200 mL) was added and the supernatant filtered through a silicagel bed. The solvent was removed from the filtrate and resultant oildissolved in hexane. The suspension was filtered through a silica gelplug and the solvent removed, yielding crude9-(2′-decyltetradecanoyloxy)nonanal (5 g) as an oil.

A solution of the product (5 g), acetic acid (0.45 g) and2-N,N-dimethylaminoethylamine (0.32 g) in methylene chloride (20 mL) wastreated with sodium triacetoxyborohydride (1.6 g) for two hours. Thesolution was washed with aqueous sodium hydroxide solution. The organicphase was dried over anhydrous magnesium sulfate, filtered and thesolvent removed. The residue was passed down a silica gel (50 g) columnusing an acetic acid/methanol/methylene chloride (2-0%/0-12%/98-88%)gradient. The purified fractions were washed with aqueous sodiumhydrogen carbonate, dried over magnesium sulphate, filtered and thesolvent removed, yielding compound 41 as an oil (2.2 g).

Example 42 Synthesis of PEG Lipid

Pegylated lipid 42-6 (“PEG-DMA”) was prepared according to the abovereaction scheme, wherein n approximates the center of the range ofethylene oxide repeating units in the pegylated lipid.

Synthesis of 42-1 and 42-2

To a solution of myristic acid (6 g, 26 mmol) in toluene (50 mL) wasadded oxalyl chloride (39 mmol, 1.5 eq. 5 g) at RT. After the resultingmixture was heated at 70° C. for 2 h, the mixture was concentrated. Theresidue was taken up in toluene and concentrated again. The residual oilwas added via a syringe to a concentrated ammonia solution (20 mL) at10° C. The reaction mixture was filtered and washed with water. Thewhite solid was dried in vacuo. The desired product was obtained as awhite solid (3.47 g, 15 mmol, 58.7%).

Synthesis of 42-3

To suspension of 20-2 (3.47 g, 15 mmol) in THF (70 mL) was added inportions lithium aluminium hydride (1.14 g, 30 mmol) at RT during 30 minperiod of time. Then the mixture was heated to reflux gently (oil bathat 65° C.) overnight. The mixture was cooled to 5 C and sodium sulphate9 hydrate was added. The mixture was stirred for 2 h, filtered through alayer of celite, washed with 15% of MeOH in DCM (200 mL). The filtrateand washings were combined and concentrated. The residual solid wasdried in vacuo. The desired product was obtained as a white solid (2.8613.4 mmol, 89.5%).

Synthesis of 42-4

To a solution of myristic acid (3.86 g, 16.9 mmol) in benzene (40 mL)and DMF (1 drop) was added oxalyl chloride (25.35 mmol, 1.5 eq. 3.22 g)at RT. The mixture was stirred at RT for 1.5 h. Heated at 60° C. for 30min. The mixture was concentrated. The residue was taken up in tolueneand concentrated again. The residual oil (light yellow) was taken in 20mL of benzene and added via syringe to a solution of 20-3 (2.86 13.4mmol) and triethylamine (3.53 mL, 1.5 eq) in benzene (40 mL) at 10° C.After addition, the resulting mixture was stirred at RT overnight. Thereaction mixture was diluted with water and was adjusted to pH 6-7 with20% H₂SO₄. The mixture was filtered and washed with water. A pale solidwas obtained. The crude product was recrystalized from methanol. Thisgave the desired product as an off-white solid (5.65 g, 13 mmol, 100%).

Synthesis of 42-5

To suspension of 20-4 (5.65 g, 13 mmol) in THF (60 mL) was added inportions lithium aluminium hydride (0.99 g, 26 mmol) at RT during 30 minperiod of time. Then the mixture was heated to reflux gently overnight.The mixture was cooled to 0 C and sodium sulphate 9 hydrate. The mixturewas stirred for 2 h, then filtered through a pad of celite and silicagel and washed with ether first. The filtrate turned cloudy andprecipitation formed. Filtration gave a white solid. The solid wasrecrystalized from MeOH and a colorless crystalline solid (2.43 g).

The pad of celite and silica gel was then washed 5% of MeOH in DCM (400mL) and then 10% of MeOH in DCM with 1% of triethylamine (300 mL). Thefractions containing the desired product were combined and concentrated.A white solid was obtained. The solid was recrystalized from MeOH and acolorless crystalline solid (0.79 g). The above two solids (2.43 g and0.79 g) were combined and dried in vacuo (3.20 g, 60%). 1HNMR (CDCl3 at7.27 ppm) δ: 2.58 (t-like, 7.2 Hz, 4H), 1.52-1.44 (m, 4H), 1.33-1.24 (m,44H), 0.89 (t-like, 6.6 Hz, 6H), 2.1-1.3 (very broad, 1H).

Synthesis of 42-6

To a solution of 20-5 (7 mmol, 2.87 g) and triethylamine (30 mmol, 4.18mL) in DCM (100 mL) was added a solution of mPEG-NHS (from NOF, 5.0mmol, 9.97 g, PEG MW approx. 2,000, n=about 45) in DCM (120 mL,). After24 h the reaction solution was washed with water (300 mL). The aqueousphase was extracted twice with DCM (100 mL×2). DCM extracts werecombined, washed with brine (100 mL). The organic phase was dried oversodium sulfate, filtered, concentrated partially. The concentratedsolution (ca 300 mL) was cooled at ca −15 C. Filtration gave a whitesolid (1.030 g, the unreacted starting amine). To the filtration wasadded Et3N (1.6 mmol, 0.222 mL, 4 eq) and acetic anhydride (1.6 mmol,164 mg). The mixture was stirred at RT for 3 h and then concentrated toa solid. The residual solid was purified by column chromatography onsilica gel (0-8% methanol in DCM). This gave the desired product as awhite solid (9.211 g). 1HNMR (CDCl3 at 7.27 ppm) δ: 4.19 (s, 2H),3.83-3.45 (m, 180-200H), 3.38 (s, 3H), 3.28 (t-like, 7.6 Hz, 2H, CH₂N),3.18 (t-like, 7.8 Hz, 2H, CH2N), 1.89 (s, 6.6H, water), 1.58-1.48 (m,4H), 1.36-1.21 (m, 48-50H), 0.88 (t-like, 6.6 Hz, 6H).

Example 43 Synthesis of PEG Lipid

A suspension of palmitic acid (10 g) in benzene (50 mL) was treated withoxalyl chloride (5 mL) for three hours. The solvent was removed and theresidue dissolved in dichloromethane (40 mL). The solution was slowlyadded to concentrated ammonia (100 mL) with stirring. The resultantsuspension was filtered and washed with water. The precipitate wassuspended in methanol, warmed to 50 C to dissolve the solid and thencooled to room temperature. The recrystallized crude product wasfiltered and dried, yielding a hexadecanoylamide as a white solid (8.7g).

The crude product was suspended in THF (50 mL) and treated with lithiumaluminum hydride (1.1 g, added slowly). The reaction mixture was stirredfor an hour. Excess methanol was added slowly, followed by water (2 mL).Dichloromethane (200 mL) was added and the mixture filtered. The solventwas removed from the filtrate, yielded crude hexadecylamine (7 g)

A solution of hexadecyanoyl chloride (10.5 g) prepared as above, indichlormethane (40 mL) was added slowly to a solution of the crudehexdecylamine in dichlormethane (40 mL) with stirring. Triethylamine (15mL) was added and the solution stirred at room temperature overnight.The solution was filtered and the collected precipitate(N-(hexadecanoyl)hexadecylamide, approximately 10.7 g) dried undervacuum.

The crude product was suspended in THF (60 mL) and treated with lithiumaluminum hydride (1.1 g, added slowly). The reaction mixture was stirredat room temperature overnight. Excess methanol was added slowly,followed by water (3 mL). Dichloromethane (250 mL) was added and thesolution filtered. The solvent was removed, yielding crudedihexadecylamine (7.6 g) as a white powder.

A solution of dihexadecylamine (3 g) and monomethoxy-PEG-2000-acetoylN-hydroxysuccinimide ester (10 g) in dichloromethane (60 mL) was treatedwith triethylamine (3 mL) and stirred overnight. Acetic anhydride (1 mL)was added and the solution stirred for 30 minutes. The reaction mixturewas diluted with dichloromethane and washed with brine. The organicfraction was dried over magnesium sulfate, filtered and the solventremoved. The residue was passed down silica gel (75 g) columns using a0-8% Methanol/dichloromethane gradient to yield 43-1 as a white powder(5.2 g).

Example 44 Synthesis of PEG Lipid

A suspension of stearic acid (6.5 g) in benzene (20 mL) was treated withoxalyl chloride (7 mL) for three days. The solvent was removed and theresidue dissolved in benzene (40 mL). Concentrated ammonia (50 mL) wasslowly added with stirring. The resultant suspension was filtered andwashed with water. The precipitate was suspended in methanol andfiltered. The collected precipitate was washed with methanol and dried,yielding a octadecanoylamide as a white powder (6.5 g).

The crude product was suspended in THF (80 mL) and treated with excesslithium aluminum hydride (1.6 g, added slowly). The reaction mixture wasstirred for an hour. Excess methanol was added slowly, followed byconcentrated hydrochloric acid until the precipitate dissolved. Thereaction mixture was diluted with water and allowed to recrystallize.The solution was filtered and the collected precipitate dried, yieldedcrude octadecylamine (5.9 g)

A solution of octadecyanoyl chloride (8 g) prepared as above, indichloromethane (40 mL) was treated with the crude octadecylamine withstirring. Triethylamine (15 mL) and hexane (100 mL) were added and thesolution stirred at 45 C for an hour. The solution was cooled, filteredand the collected precipitate washed with methanol. The precipitate wasrecrystallized from dichloromethane, yieldingN-(octadecanoyl)octadecylamide (6.8 g) as a white powder.

The product was suspended in THF (150 mL) and treated with lithiumaluminum hydride (1.5 g, added slowly). The reaction mixture wasrefluxed overnight. Excess methanol was added slowly, followed byconcentrated hydrochloric acid until the precipitate dissolved. Thesolution was diluted with water and allowed to crystallize. The solutionwas filtered and the collected precipitate washed with water (4×). Theprecipitate was dried under vacuum, yielding dioctadecylamine (6.2 g) asa white powder.

A solution of dioctadecylamine (4.5 g) and monomethoxy-PEG-2000-acetoylN-hydroxysuccinimide ester (9 g) in chloroform (90 mL) was treated withtriethylamine (40 mL) and stirred at 50 C for 30 minutes. The solutionwas filtered. Acetic anhydride (1 mL) was added to the filtrate and thesolution stirred for 15 minutes. Ammonia (150 mL) was added, followed bybrine (150 mL). The reaction mixture was extracted with dichloromethaneand the organic phase washed with dilute hydrochloric acid. The organicfraction was dried over magnesium sulfate, filtered and the solventremoved. The residue was passed down silica gel (75 g) columns using a0-8% Methanol/dichloromethane gradient to yield 44-1 as a white powder(7.4 g).

Example 45 Synthesis of PEG Lipid

A suspension of lauric acid (10 g) in benzene (20 mL) was treated withoxalyl chloride (10 mL) for an hour. The solvent was removed and theresidue dissolved in dichloromethane (50 mL). The solution was slowlyadded to concentrated ammonia (150 mL) with stirring. The reactionmixture was washed with dichloromethane. The organic phase was driedover magnesium sulfate, filtered and the solvent removed, yielding crudedodecanoylamide as a white powder (10 g).

The product was suspended in THF (150 mL) and treated with lithiumaluminum hydride (3 g, added slowly). The reaction mixture was stirredfor an hour. Excess methanol was added slowly, followed by aqueoussodium hydroxide solution (5 mL). Dichloromethane (100 mL) was added andthe resultant suspension filtered. The solvent was removed from thefiltrate, and the residue passed down a silica gel (80 g) column using amethanol/dichloromethane gradient, yielding dodecanylamine (4.5 g).

A solution of lauric acid (7.3 g), crude tetradecylamine (4.5 g) andN-hydroxysuccinimide (4.2 g) in dichloromethane (200 mL) was treatedwith EDC (7.0 g) followed by triethylamine (15 mL), and the solutionstirred at room temperature overnight. The solution was filteredyielding crude N-dodecanoyldodecanylamide (1 g) as a powder. The solventwas removed from the filtrate, the residue suspended in methanol andfiltered, yielding a further 2 g of crude N-dodecanoyldodecanylamide.

The crude product (3 g) was suspended in THF (60 mL) and treated withlithium aluminum hydride (excess, added slowly) for two hours. Excessmethanol was added slowly, followed by water (1 mL). Dichloromethane(100 mL) was added and the suspension filtered. The solvent was removedfrom the filtrate and the residue passed down a silica gel column (20 g)using a 0-12% methanol/dichloromethane gradient yieldingN-dodecanoyldodecanylamine (1.4 g) as a waxy solid.

A solution of N-dodecanyldodecanylamine (0.52 g) andmonomethoxy-PEG-2000-acetoyl N-hydroxysuccinimide ester (1.5 g) indichloromethane (10 mL) was treated with triethylamine (0.2 mL) andstirred overnight. The solvent was removed and the product passed down asilica gel column (20 g) using a 0-6% methanol/dichloromethane gradient.Acetic anhydride (5 drops) and trimethylamine (10 drops) was added to asolution of the recovered product in dichloromethane and allowed to stirfor an hour. The solvent removed and the residue was passed down asilica gel (20 g) column using a 0-4% methanol/dichloromethane gradientto yield MePEGA-2000-DLA as a white powder (0.44 g).

Example 46 Synthesis of PEG Lipid

A suspension of myristic acid (30 g) in benzene (100 mL) was treatedwith oxalyl chloride (15 mL) overnight. The solvent was removed and theresidue dissolved in dichloromethane (100 mL). The solution was slowlyadded to concentrated ammonia (70 mL) with stirring. The resultantsuspension was filtered and washed with water. The precipitate wasdried, yielding crude tetradecanoylamide as a white solid (27 g).

The product was suspended in THF (200 mL) and treated with lithiumaluminum hydride (4.5 g, added slowly). The reaction mixture was stirredfor an hour. Excess methanol was added slowly, followed by water (10mL). Dichloromethane (250 mL) was added and the resultant suspensionfiltered. The solvent was removed from the filtrate, yielded crudetetradecylamine (17.6 g).

A solution of lauric acid (3.5 g), crude tetradecylamine (3 g) andN-hydroxysuccinimide (1.9 g) in dichloromethane (40 mL) was treated withEDC (3.3 g) followed by triethylamine (4 mL), and the solution stirredat room temperature for three days. The solution was filtered and thecollected precipitate dried, yielding crude N-lauroyltetradecanylamine(2.6 g) as a powder.

The crude product was suspended in THF (60 mL) and treated with lithiumaluminum hydride (0.8 g, added slowly) for one hour. Excess methanol wasadded slowly, followed by water (2 mL). Dichloromethane was added andthe suspension filtered. The solvent was removed and the residuedissolved in hot methanol (100 mL). The solution was cooled and filteredto yield 0.5 g of N-dodecanyltetradecanylamine. The solvent was removedfrom the filtrate and the process repeated with 20 mL methanol toproduce a second crop of N-dodecanyltetradecanylamine (0.9 g)

A solution of N-dodecanyltetradecanylamine (0.5 g) andmonomethoxy-PEG-2000-acetoyl N-hydroxysuccinimide ester (1.5 g) indichloromethane (10 mL) was treated with triethylamine (0.2 mL) andstirred overnight. The solvent was removed and the product passed down asilica gel column (20 g) using a 0-6% methanol/dichloromethane gradient.Acetic anhydride (5 drops) and trimethylamine (10 drops) was added to asolution of the recovered product in dichloromethane and allowed to stirfor an hour. The solvent removed and the residue was passed down asilica gel (20 g) column using a 0-4% methanol/dichloromethane gradientto yield MePEGA-2000-LMA as a white powder (0.76 g).

Example 47 Luciferase mRNA In Vivo Evaluation Using the LipidNanoparticle Compositions

Cationic lipid (MC3), DSPC, cholesterol and PEG-lipid were solubilizedin ethanol at a molar ratio of 50:10:38.5:1.5. Lipid nanoparticles (LNP)were prepared at a total lipid to mRNA weight ratio of approximately10:1 to 30:1. Briefly, the mRNA was diluted to 0.2 mg/mL in 10 to 50 mMcitrate buffer, pH 4. Syringe pumps were used to mix the ethanolic lipidsolution with the mRNA aqueous solution at a ratio of about 1:5 to 1:3(vol/vol) with total flow rates above 15 ml/min. The ethanol was thenremoved and the external buffer replaced with PBS by dialysis. Finally,the lipid nanoparticles were filtered through a 0.2 μm pore sterilefilter. Lipid nanoparticle particle size was 70-90 nm diameter asdetermined by quasi-elastic light scattering using a Nicomp 370submicron particle sizer (Santa Barbara, Calif.).

Studies were performed in 6-8 week old female C57BL/6 mice (CharlesRiver) according to guidelines established by an institutional animalcare committee (ACC) and the Canadian Council on Animal Care (CCAC).Varying doses of mRNA-lipid nanoparticle were systemically administeredby tail vein injection and animals euthanized at specific time points(1, 2, 4, 8 and 24 hrs) post-administration. Liver and spleen werecollected in pre-weighted tubes, weights determined, immediately snapfrozen in liquid nitrogen and stored at −80° C. until processing foranalysis.

For liver, approximately 50 mg was dissected for analyses in a 2 mLFastPrep tubes (MP Biomedicals, Solon Ohio). ¼″ ceramic sphere (MPBiomedicals) was added to each tube and 500 μL of Glo Lysis Buffer—GLB(Promega, Madison Wis.) equilibrated to room temperature was added toliver tissue. Liver tissues were homogenized with the FastPrep24instrument (MP Biomedicals) at 2×6.0 m/s for 15 seconds. Homogenate wasincubated at room temperature for 5 minutes prior to a 1:4 dilution inGLB and assessed using SteadyGlo Luciferase assay system (Promega).Specifically, 50 μL of diluted tissue homogenate was reacted with 50 μLof SteadyGlo substrate, shaken for 10 seconds followed by 5 minuteincubation and then quantitated using a CentroXS³ LB 960 luminometer(Berthold Technologies, Germany). The amount of protein assayed wasdetermined by using the BCA protein assay kit (Pierce, Rockford Ill.).Relative luminescence units (RLU) were then normalized to total ugprotein assayed. To convert RLU to ng luciferase a standard curve wasgenerated with QuantiLum Recombinant Luciferase (Promega). Based in thedata provided in FIG. 1, the four-hour time point was chosen forefficacy evaluation of the lipid formulations (see EXAMPLE 48).

The FLuc mRNA (L-6107) from Trilink Biotechnologies will express aluciferase protein, originally isolated from the firefly, Photinuspyralis. FLuc is commonly used in mammalian cell culture to measure bothgene expression and cell viability. It emits bioluminescence in thepresence of the substrate, luciferin. This capped and polyadenylatedmRNA is fully substituted with 5-methylcytidine and pseudouridine.

Example 48 Determination of Efficacy of Lipid Nanoparticle FormulationsContaining Various Cationic Lipids Using an In Vivo Luciferase mRNAExpression Rodent Model

The cationic lipids shown in Table 2 have previously been tested withnucleic acids. For comparative purposes, these lipids were also used toformulate lipid nanoparticles containing the FLuc mRNA (L-6107) using anin line mixing method, as described in EXAMPLE 47 and in PCT/US10/22614,which is hereby incorporated by reference in its entirety. Lipidnanoparticles were formulated using the following molar ratio: 50%Cationic lipid/10% distearoylphosphatidylcholine (DSPC)/38.5%Cholesterol/1.5% PEG lipid (“PEG-DMG”, i.e.,(1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol, with anaverage PEG molecular weight of 2000). Relative activity was determinedby measuring luciferase expression in the liver 4 hours followingadministration via tail vein injection as described in EXAMPLE 47. Theactivity was compared at a dose of 0.3 and 1.0 mg mRNA/kg and expressedas ng luciferase/g liver measured 4 hours after administration, asdescribed in EXAMPLE 47.

TABLE 2 Lipids showing activity with mRNA Liver Luc Liver Luc @ 0.3 mg/@ 1.0 mg/ Compound kg dose kg dose Structure MC2 4 ± 1 N/D

DLinDMA 13 ± 3  67 ± 20

MC4 41 ± 10 N/D

XTC2 80 ± 28 237 ± 99 

MC3 198 ± 126 757 ± 528

319 (2% PEG) 258 ± 67  681 ± 203

137 281 ± 203 588 ± 303

The novel lipids of the invention shown in Table 3 were formulated usingthe following molar ratio: 50% cationic lipid/10%distearoylphosphatidylcholine (DSPC)/38.5% Cholesterol/1.5% PEG lipid(“PEG-DMA” compound 42-6). Relative activity was determined by measuringluciferase expression in the liver 4 hours following administration viatail vein injection as described in EXAMPLE 47. The activity wascompared at a dose of 0.3 and 1.0 mg mRNA/kg and expressed as ngluciferase/g liver measured 4 hours after administration, as describedin EXAMPLE 47.

TABLE 3 Novel Cationic lipids Liver Luc Liver Luc @ 0.3 @ 1.0 mg/kgmg/kg (ng luc/g (ng luc/g No. pK_(a) liver) liver) Structure 2 5.64 23 ±6  84 ± 24

3 7.15 1.5 ± 0.6 0.32 ± 0.1 

4 6.43 53 ± 10 126 ± 102

5 6.28 1571 ± 414  12900 ± 4083

6 6.12 2015 ± 135  16006 ± 2487

11 6.36 328 ± 12  1266 ± 522 

13 6.51 16 ± 4  20 ± 20

15 6.30 66 ± 32 243 ± 71 

16 6.63 7 ± 1 14 ± 4 

19 6.72 8 ± 3 15 ± 21

20 6.44 301 ± 155 501 ± 96 

21 6.28 404 ± 103 4149 ± 2514

22 6.53 2126 ± 294  14486 ± 6607 

23 6.24 1109 ± 209  4568 ± 762 

24 6.28 1094 ± 180  6928 ± 1015

25 6.20 901 ± 173 6223 ± 1714

26 6.89 30 ± 5   55 ± 0.8

27 6.30 724 ± 144 6636 ± 1230

28 6.20 522 ± 86  4582 ± 1291

29 6.22 1303 ± 445  9750 ± 3519

31 6.33 344 ± 160 4103 ± 1073

32 6.47 626 ± 299 12266 ± 192  

33 6.27 711 ± 272 4448 ± 512 

35 6.21 35 ± 15 101 ± 69 

38 6.24 977 ± 336 7135 ± 1020

39 5.82 36 ± 13 326 ± 81

40 6.38 236 ± 71  1704 ± 571 

41 5.91 63 ± 27 799 ± 619

Example 49 Determination of pK_(A) of Formulated Lipids

As described elsewhere, the pKa of formulated cationic lipids iscorrelated with the effectiveness of LNPs for delivery of nucleic acids(see Jayaraman et al, Angewandte Chemie, International Edition (2012),51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176(2010)). The preferred range of pKa is ˜5 to ˜7. The pK_(a) of eachcationic lipid was determined in lipid nanoparticles using an assaybased on fluorescence of 2-(p-toluidino)-6-napthalene sulfonic acid(TNS). Lipid nanoparticles comprising of cationiclipid/DSPC/cholesterol/PEG-lipid (50/10/38.5/1.5 mol %) in PBS at aconcentration of 0.4 mM total lipid are prepared using the in-lineprocess as described in EXAMPLE 47. TNS was prepared as a 100 μM stocksolution in distilled water. Vesicles were diluted to 24 μM lipid in 2mL of buffered solutions containing, 10 mM HEPES, 10 mM MES, 10 mMammonium acetate, 130 mM NaCl, where the pH ranged from 2.5 to 11. Analiquot of the TNS solution was added to give a final concentration of 1μM and following vortex mixing fluorescence intensity was measured atroom temperature in a SLM Aminco Series 2 Luminescence Spectrophotometerusing excitation and emission wavelengths of 321 nm and 445 nm. Asigmoidal best fit analysis was applied to the fluorescence data and thepK_(a) was measured as the pH giving rise to half-maximal fluorescenceintensity (see FIG. 2).

Example 50 Comparative Activity of Amino Lipids and the Effect ofHydrocarbon Chain Structure

The cationic lipids shown in Table 5 were formulated using the followingmolar ratio: 50% Cationic lipid/10% distearoylphosphatidylcholine(DSPC)/38.5% Cholesterol/1.5% PEG-lipid (42-6). Relative activity wasdetermined by measuring luciferase expression in the liver 4 hoursfollowing administration via tail vein injection as described in EXAMPLE48. The activity was compared at a dose of 0.1, 0.3 and 1.0 mg mRNA/kgand expressed as ng luciferase/g liver measured 4 hours afteradministration, as described in EXAMPLE 48. Data is plotted in FIG. 3(from top to bottom: diamond=compound 6; square=compound 5;triangle=MC3; and circle=compound A). Compounds A, 5 and 6 have a commonheadgroup but different hydrocarbon chain structures.

TABLE 5 Comparative Data Liver Luc Liver Luc @ 0.3 mg/kg @ 1.0 mg/kgCompound pKa (ng luc/g liver) (ng luc/g liver) Structure MC3 6.09 603 ±150 2876 ± 622 

A 6.26 77 ± 40 203 ± 122

5 6.28 1571 ± 414  12900 ± 4083

6 6.12 2015 ± 135 16006 ± 2487

Example 51 Comparative Activity of Cationic Lipids and the Effect ofHeadgroup Chain Length

The cationic lipids shown in Table 6 were formulated using the followingmolar ratio: 50% Cationic lipid/10% distearoylphosphatidylcholine(DSPC)/38.5% Cholesterol/1.5% PEG-lipid (42-6). Relative activity wasdetermined by measuring luciferase expression in the liver 4 hoursfollowing administration via tail vein injection as described in EXAMPLE48. The activity was compared at a dose of 0.1, 0.3 and 1.0 mg mRNA/kgand expressed as ng luciferase/g liver measured 4 hours afteradministration, as described in EXAMPLE 48. Compounds A, B and C havecommon hydrocarbon chain structure but different headgroup chain length.Compound 6 shares a preferred headgroup with compound A and demonstratesthe unexpected advantage of the combination of headgroup and hydrocarbonchain structure.

TABLE 6 Comparative Data Liver Luc Liver Luc @ 0.3 mg/kg @ 1.0 mg/kgCompound pKa (ng luc/g liver) (ng luc/g liver) Structure A 6.26 77 ± 40203 ± 122

B 6.68   3 ± 0.8 12 ± 4 

C 7.32   2 ± 0.5 7 ± 2

6 6.12 2015 ± 135  16006 ± 2487 

Example 52 Comparative Activity of PEG-DMG and PEG-DMA Lipids

The comparative activity of PEG-DMG and PEG-DMA lipids is shown in FIG.4. LNPs were formulated using the following molar ratio: 50% MC3lipid/10% distearoylphosphatidylcholine (DSPC)/38.5% Cholesterol/1.5%PEG-lipid (PEG-DMG or PEG-DMA). Relative activity was determined bymeasuring luciferase expression in the liver 4 hours followingadministration via tail vein injection as described in EXAMPLE 48. Theactivity was compared at a dose of 0.1, 0.3 and 1.0 mg mRNA/kg andexpressed as ng luciferase/g liver measured 4 hours afteradministration, as described in EXAMPLE 48.

Data is presented in bar graph form in FIG. 4.

Example 53 LNP Activity Using a Red Fluorescent Protein Called CherryRed (CR) mRNA

LNPs using compound 6 are formulated as described in EXAMPLE 47 withmRNA coding for a red fluorescent protein called cherry red (CR, e.g.TriLink Biotechnologies product L-6113). In vivo studies are conductedas described in EXAMPLE 47 and sections of liver tissue are processedand observed by confocal fluorescence microscopy at 4, 6 and 24 hourspost administration. Expression levels peaked at around 6 hours andmaintained to at least 24 hours. The observed tissue sectionsdemonstrate homogenous expression throughout the liver.

Example 54 LNP Delivery of Human Fix mRNA to Hepatocytes In Vivo Resultsin Therapeutic Levels of hFIX Protein in Mouse Plasma

The LNPs with cationic lipids shown in Table 7 are formulated with mRNAcoding for human FIX (e.g. TriLink Biotechnologies product L-6110) usingthe following molar ratio of lipids: 50% lipid/10%distearoylphosphatidylcholine (DSPC)/38.5% Cholesterol/1.5% PEG-lipid asdescribed in EXAMPLE 47. Plasma protein is analyzed by ELISA using acommercially available kit (e.g. Abcam ab108831) as per the suppliersinstructions. The measured levels of hFIX expression given in Table 7for LNPs of compound 5 and compound 6 are clinically relevant andadministering these hFIX mRNA LNPs at a dose of 1 mg/kg results in hFIXprotein concentrations that would be sufficient to move patients withsevere disease into mild disease status. The duration of the hFIX atthese levels is ˜15 hours or longer.

TABLE 7 Comparative Data Human Factor IX Human Factor IX @ 0.3 mg/kg (ng@ 1.0 mg/kg (ng Compound mL in plasma) mL in plasma) Structure MC3 336 ±21  1154 ± 25  

5 1097 ± 357  4556 ± 686 

6 713 ± 148 2697 ± 345 

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments. These and other changes can be made to the embodiments inlight of the above-detailed description. In general, in the followingclaims, the terms used should not be construed to limit the claims tothe specific embodiments disclosed in the specification and the claims,but should be construed to include all possible embodiments along withthe full scope of equivalents to which such claims are entitled.Accordingly, the claims are not limited by the disclosure.

The invention claimed is:
 1. A pegylated lipid having the followingstructure (II):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein: R¹⁰ and R¹¹ are each independently a straight or branched,saturated or unsaturated alkyl chain containing from 10 to 30 carbonatoms, wherein the alkyl chain is optionally interrupted by one or moreester bonds; and z has a mean value ranging from 30 to 60; provided thatR¹⁰ and R¹¹ are not both n-octadecyl when z is
 42. 2. The pegylatedlipid of claim 1, wherein R¹⁰ and R¹¹ are each independently straight,saturated alkyl chains containing from 12 to 16 carbon atoms.
 3. Thepegylated lipid of claim 1, wherein z is about
 45. 4. The pegylatedlipid of claim 1, having one of the following structures:

wherein n has a mean value ranging from 40 to
 50. 5. A compositioncomprising the pegylated lipid of claim 1 and a cationic lipid.
 6. Thecomposition of claim 5, wherein the cationic lipid has a structure ofFormula I:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein: L¹ and L² are each independently —O(C═O)—, —(C═O)O— ora carbon-carbon double bond; R^(1a) and R^(1b) are, at each occurrence,independently either (a) H or C₁-C₁₂ alkyl, or (b) R^(1a) is H or C₁-C₁₂alkyl, and R^(1b) together with the carbon atom to which it is bound istaken together with an adjacent R^(1b) and the carbon atom to which itis bound to form a carbon-carbon double bond; R^(2a) and R^(2b) are, ateach occurrence, independently either (a) H or C₁-C₁₂ alkyl, or (b)R^(2a) is H or C₁-C₁₂ alkyl, and R^(2b) together with the carbon atom towhich it is bound is taken together with an adjacent R^(2b) and thecarbon atom to which it is bound to form a carbon-carbon double bond,R^(3a) and R^(3b) are, at each occurrence, independently either (a) H orC₁-C₁₂ alkyl, or (b) R^(3a) is H or C₁-C₁₂ alkyl, and R^(3b) togetherwith the carbon atom to which it is bound is taken together with anadjacent R^(3b) and the carbon atom to which it is bound to form acarbon-carbon double bond; R^(4a) and R^(4b) are, at each occurrence,independently either (a) H or C₁-C₁₂ alkyl, or (b) R^(4a) is H or C₁-C₁₂alkyl, and R^(4b) together with the carbon atom to which it is bound istaken together with an adjacent R^(4b) and the carbon atom to which itis bound to form a carbon-carbon double bond; R⁵ and R⁶ are eachindependently methyl or cycloalkyl; R⁷ is, at each occurrence,independently H or C₁-C₁₂ alkyl; R⁸ and R⁹ are each independentlyunsubstituted C₁-C₁₂ alkyl; or R⁸ and R⁹, together with the nitrogenatom to which they are attached, form a 5, 6 or 7-membered heterocyclicring comprising one nitrogen atom; a and d are each independently aninteger from 0 to 24; b and c are each independently an integer from 1to 24; and e is 1 or
 2. 7. The composition of claim 6, wherein one of L¹or L² is —O(C═O)—.
 8. The composition of claim 6, wherein one of L¹ orL² is —(C═O)O—.
 9. The composition of claim 6, wherein the cationiclipid has one of the following structures (Ia), (Ib) or (Ic):


10. The composition of claim 9, wherein a, b, c and d are eachindependently an integer from 2 to
 12. 11. The composition of claim 9,wherein a, b, c and d are each independently an integer from 5 to
 9. 12.The composition of claim 9, wherein at least one of R^(1a), R^(2a),R^(3a) and R^(4a) is H.
 13. The composition of claim 9, wherein at leastone of R^(1a), R^(2a), R^(3a) and R^(4a) is C₁-C₈ alkyl.
 14. Thecomposition of claim 13, wherein C₁-C₈ alkyl is methyl, ethyl, n-propyl,iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.
 15. Thecomposition of claim 9, wherein each of R⁵ and R⁶ is methyl.
 16. Thecomposition of claim 9, wherein e is
 2. 17. The composition of claim 9,wherein each of R⁸ and R⁹ is methyl.
 18. The composition of claim 5,wherein the cationic lipid has one of the following structures:


19. The composition of claim 5, further comprising a nucleic acid. 20.The composition of claim 19, wherein the nucleic acid is selected fromantisense and messenger RNA.
 21. The composition of claim 5, furthercomprising one or more excipient selected from neutral lipids andsteroids.
 22. The composition of claim 21, wherein the compositioncomprises one or more neutral lipids selected from DSPC, DPPC, DMPC,DOPC, POPC, DOPE and SM.
 23. The composition of claim 22, wherein theneutral lipid is DSPC.
 24. The composition of claim 21, wherein thesteroid is cholesterol.
 25. The pegylated lipid of claim 1, wherein R¹⁰and R¹¹ are each independently a straight or branched, saturated orunsaturated alkyl chain containing 12 carbon atoms.
 26. The pegylatedlipid of claim 1, wherein R¹⁰ and R¹¹ are each independently a straightor branched, saturated or unsaturated alkyl chain containing 14 carbonatoms.
 27. The pegylated lipid of claim 1, wherein R¹⁰ and R¹¹ are eachindependently a straight or branched, saturated or unsaturated alkylchain containing 16 carbon atoms.